Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

• (40)Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., Segner, H (2007), ‘Concentration- and time-dependent effects of the synthetic estrogen, 17 alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio’ Journal of Toxicology and Environmental Health-Part A, 70, 9-10 pp 768-779.

• (41)OECD (2011), Validation Report (Phase 1) for the Fish Sexual Development Test, Series on Testing and Assessment No 141, ENV/JM/MONO(2011)22, OECD, Paris.

• (42)OECD (2011), Validation Report ( Phase 2) for the Fish Sexual Development Test, Series on Testing and Assessment No 142, ENV/JM/MONO(2011)23, OECD, Paris.

• (43)OECD (2011), Peer Review Report of the validation of the Fish Sexual Development Test, Series on Testing and Assessment No 143, ENV/JM/MONO(2011)24, OECD, Paris.

• (44)Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. OJ L 276, 20.10.2010, p. 33.

Appendix 1

Abbreviations and definitions

Apical endpoint : Causing effect at population level

ASV : Air saturation value

Biomarker : Causing effect at individual level

Chemical : A substance or a mixture.

Dph : Days post hatch

DMY : Y-specific DM-domain gene required for male development in the medaka fish

ELISA : Enzyme-Linked Immunosorbent Assay

Fish weight : Fish wet weight (blotted dry)

FSDT : Fish Sexual Development Test

HPG axis : Hypothalamic-pituitary-gonadal axis

Intersex fish : Fish with more than one oocyte in testis per 6 sections analysed or spermatogenetic cells in ovaries (yes/no)

Loading rate : Wet weight of fish per volume of water

MOA : Mode of action

RT-PCR : Reverse Transcriptase Polymerase Chain-Reaction

Test chemical : Any substance or mixture tested using this test method.

Undifferentiated fish : Fish with gonads exhibiting no discernible germ cells.

VTG : Vitellogenin

Appendix 2

Experimental conditions for the FSDT (freshwater species)

1. Recommended species	Japanese medaka (Oryzias latipes)	Zebrafish (Danio rerio)	Three-spined Stickleback (Gasterostreus aculeatus)
2. Test type	Flow-through or semi-static	Flow-through or semi-static	Flow-through or semi-static
3. Water temperature	25 ± 2 °C	27 ± 2 °C	20 ± 2 °C
4. Illumination quality	Fluorescent bulbs (wide spectrum)	Fluorescent bulbs (wide spectrum)	Fluorescent bulbs (wide spectrum
5. Light intensity	10-20 μE/m2/s, 540-1 080 lux, or 50-100 ft-c (ambient laboratory levels)	10-20 μE/m2/s, 540-1 080 lux, or 50-100 ft-c (ambient laboratory levels)	10-20 μE/m2/s, 540-1 080 lux, or 50-100 ft-c (ambient laboratory levels)
6. Photoperiod	12-16 h light, 8-12 h dark	12-16 h light, 8-12 h dark	16 h light, 8 h dark
7. Minimum chamber size	Individual chambers should contain a minimum of 7 l water volume	Individual chambers should contain a minimum of 7 l water volume	Individual chambers should contain a minimum of 7 l water volume
8. Volume exchanges of test solutions	Minimum of 5 daily	Minimum of 5 daily	Minimum of 5 daily
9. Age of test organisms at start of exposure	Newly fertilised eggs (Early blastula stage)	Newly fertilised eggs (Early blastula stage)	Newly fertilised eggs
10. No. of eggs per treatment	Minimum 120	Minimum 120	Minimum 120
11. No. of treatments	Minimum 3 (plus appropriate controls)	Minimum 3 (plus appropriate controls)	Minimum 3 (plus appropriate controls)
12. No. replicates per treatment	Minimum 4 (unless square root allocation to controls)	Minimum 4 (unless square root allocation to controls)	Minimum 4 (unless square root allocation to controls)
13. Feeding regime	Live Artemia, frozen adult brine shrimp, flake food, etc. It is recommended to feed twice daily	Special fry food, live Artemia, frozen adult brine shrimp, flake food, etc. It is recommended to feed twice daily	Live Artemia, frozen adult brine shrimp, flake food, etc. It is recommended to feed twice daily
14. Aeration	None unless DO concentration falls below 60 % saturation	None unless DO concentration falls below 60 % saturation	None unless DO concentration falls below 70 % saturation
15. Dilution water	Clean surface, well or reconstituted water	Clean surface, well or reconstituted water	Clean surface, well or reconstituted water
16. Test chemical exposure duration	60-dph	60-dph	60-dph
17. Biological endpoints	Hatching success, Survival Gross- morphology, VTG gonadal histology, Genetic sex, Sex ratio	Hatching success, Survival Gross- morphology, VTG gonadal histology, Sex ratio	Hatching success, Survival Gross- morphology, VTG gonadal histology, Sex ratio
18. Test acceptability criteria for pooled replicates of controls	Hatching success > 80 %	Hatching success > 80 %	Hatching success > 80 %
18. Test acceptability criteria for pooled replicates of controls	Post hatch survival ≥ 70 %	Post hatch survival ≥ 70 %	Post hatch survival ≥ 70 %
18. Test acceptability criteria for pooled replicates of controls	growth (Fish wet weight, blotted dry) > 150 mg	growth (Fish wet weight, blotted dry) > 75 mg	growth (Fish wet weight, blotted dry) > 120 mg
18. Test acceptability criteria for pooled replicates of controls	Length (standard length) > 20mm	Length (standard length) > 14 mm	Length (standard length) > 20 mm
18. Test acceptability criteria for pooled replicates of controls	Sex ratio (% males or females) 30 %-70 %	Sex ratio (% males or females) 30 %-70 %	Sex ratio (% males or females) 30 %-70 %

Appendix 3

Chemical characteristics of an acceptable dilution water

CONSTITUENT	CONCENTRATION
Particular matter	< 20 mg/l
Total organic carbon	< 2 mg/l
Unionised ammonia	< 1 μg/l
Residual chlorine	< 10 μg/l
Total organophosphorus pesticides	< 50 ng/l
Total organochlorine pesticides plus polychlorinated biphenyls	< 50 ng/l
Total organic chlorine	< 25 ng/l

Appendix 4

From test method C.14/Guidance on test concentrations

Column (Number of concentrations between 100 and 10, or between 10 and 1) [^E0206]	Column (Number of concentrations between 100 and 10, or between 10 and 1) [^E0206]	Column (Number of concentrations between 100 and 10, or between 10 and 1) [^E0206]	Column (Number of concentrations between 100 and 10, or between 10 and 1) [^E0206]	Column (Number of concentrations between 100 and 10, or between 10 and 1) [^E0206]	Column (Number of concentrations between 100 and 10, or between 10 and 1) [^E0206]	Column (Number of concentrations between 100 and 10, or between 10 and 1) [^E0206]
1	2	3	4	5	6	7
100	100	100	100	100	100	100
32	46	56	63	68	72	75
10	22	32	40	46	52	56
3,2	10	18	25	32	37	42
1,0	4,6	10	16	22	27	32
	2,2	5,6	10	15	19	24
	1,0	3,2	6,3	10	14	18
		1,8	4,0	6,8	10	13
		1,0	2,5	4,6	7,2	10
			1,6	3,2	5,2	7,5
			1,0	2,2	3,7	5,6
				1,5	2,7	4,2
				1,0	1,9	3,2
					1,4	2,4
					1,0	1,8
						1,3
						1,0

A series of three (or more) successive concentrations may be chosen from a column. Mid-points between concentrations in column (x) are found in column (2x + 1). The values listed can represent concentrations expressed as percentage per volume or weight (mg/l or μg/l). Values can be multiplied or divided by any power of 10 as appropriate. Column 1 might be used if there was considerable uncertainty on the toxicity level.

Appendix 5

Guidance for homogenisation of head & tail from juvenile zebrafish, fathead minnow, three spined stickleback and Japanese medaka

The purpose of this section is to describe the procedures that occur prior to the quantification of the VTG concentration. Other procedures that result in comparable VTG quantification can be used. It is an option to determine the VTG concentration in blood plasma or liver instead of head/tail homogenate.

Procedure

• 1.The fish are anaesthetised and euthanised in accordance with the test description.

• 2.The head and tail are cut of the fish in accordance with the test description. Important: All dissection instruments, and the cutting board should be rinsed and cleaned properly (e.g. with 96 % ethanol) between handling of each single fish to prevent ‘VTG pollution’ from females or induced males to un-induced males.

• 3.The weight of the pooled head and tail from each fish is measured to the nearest mg.

• 4.After being weighed, the parts are placed in appropriate tubes (e.g. 1,5 ml eppendorf) and frozen at – 80 °C until homogenisation or directly homogenised on ice with two plastic pistils. (Other methods can be used if they are performed on ice and the result is a homogenous mass). Important: The tubes should be numbered properly so that the head and tail from the fish can be related to their respective body-section used for gonad histology.

• 5.When a homogenous mass is achieved an amount of 4-10 time the tissue weight of ice-cold homogenisation buffer ([^E0207]) is added (note the dilution). Keep working with the pistils until the mixture is homogeneous. Important note: New pistils are used for each fish.

• 6.The samples are placed on ice until centrifugation at 4 °C at 50 000 g for 30 min.

• 7.Use a pipette to dispense portions of 20 to 50 μl (note the amount) supernatant into at least two tubes by dipping the tip of the pipette below the fat layer on the surface and carefully sucking up the supernatant without fat- or pellet fractions.

• 8.The tubes are stored at – 80 °C until use.

Note: The homogenisation buffer should be used the same day as manufactured. Place on ice during use

Appendix 6

Guidance for quantification of head & tail homogenate vitellogenin in zebrafish (Danio rerio) (modified from Holbech et al., 2001). other procedures using homologous antibodies and standards can be used

• 1.Microtiter plates (certified Maxisorp F96, Nunc, Roskilde Denmark) previously coated with 5 μg/ml anti zebrafish lipovitellin-IgG are thawed and washed 3 times with washing buffer (*).

• 2.Purified zebrafish vitellogenin standard ([^E0208]) is serially diluted to 0,2, 0,5, 1, 2, 5, 10 and 20 ng/ml in dilution buffer (**) and samples are diluted at least 200 times (to prevent matrix effect) in dilution buffer and applied to the plates. An assay control is applied in duplicate. 150 μl are applied to each well. Standards are applied in duplicate and samples in triplicate. Incubate over night at 4 °C on a shaker.

• 3.The plates are washed 5 times with washing buffer (*)

• 4.HRP coupled to a dextran chain (e.g. AMDEX A/S, Denmark) and conjugated antibodies are diluted in washing buffer; Actual dilution differs by batch and age. 150 μl are applied to each well and the plates are incubated for 1 hour at room temperature on a shaker.

• 5.The plates are washed 5 times with washing buffer (*) and the bottom of the plates is carefully cleaned with ethanol.

• 6.150 μl TMB plus (***) are applied to each well. Protect the plate against light with tinfoil, and watch the colour development on a shaker.

• 7.When the standard curve is fully developed the enzyme activity is stopped by adding 150 μl 0,2 M H2SO4 to each well.

• 8.The absorbance is measured at 450 nm (e.g. on a Molecular Devices Thermomax plate reader). Data are analysed on the associated software (e.g. Softmax).

• (*)Washing buffer:

PBS-stock ()	500,0	ml
BSA	5,0	g
Tween 20	5,0	ml
Adjust pH to 7,3 and fill to 5 l with millipore H2O. Store at 4 °C.	Adjust pH to 7,3 and fill to 5 l with millipore H2O. Store at 4 °C.	Adjust pH to 7,3 and fill to 5 l with millipore H2O. Store at 4 °C.

• (**)Dilution buffer:

PBS-Stock ()	100,0	ml
BSA	3,0	g
Tween 20	1,0	ml
Adjust pH to 7,3 and fill to 1 l with millipore H2O. Store at 4 °C.	Adjust pH to 7,3 and fill to 1 l with millipore H2O. Store at 4 °C.	Adjust pH to 7,3 and fill to 1 l with millipore H2O. Store at 4 °C.

• (***)TMB plus is a ‘ready-to-use’ substrate produced by KemEnTec (Denmark). It is sensitive to light. Store at 4 °C.

• ()PBS stock

NaCl	160,0	g
KH2PO4	4,0	g
Na2HPO4 · 2H2O	26,6	g
KCl	4,0	g
Adjust pH to 6,8 and fill with millipore H2O to 2 l. Store at room temperature.	Adjust pH to 6,8 and fill with millipore H2O to 2 l. Store at room temperature.	Adjust pH to 6,8 and fill with millipore H2O to 2 l. Store at room temperature.

Appendix 7

Guidance for the preparation of tissue sections for sex determination and staging of gonads

The purpose of this section is to describe the procedures that occur prior to the evaluation of histological sections. Other procedures that result in similar sex determination and gonadal staging can be used.

With a few exceptions, these procedures are similar for Japanese medaka (JMD) and zebrafish (ZF).

Euthanasia, Necropsy, and Tissue Fixation

Objectives:

• 1. Provide for the humane sacrifice of fish.

• 1. Obtain necessary body weights and measurements.

• 1. Evaluate secondary sex characteristics.

• 1. Dissect tissues for VTG analysis.

• 1. Fixation of the gonads.

Procedures:

• 1. Fish should be sacrificed immediately prior to necropsy. Therefore, unless multiple prosectors are available, multiple fish should not be sacrificed simultaneously.

• 1. Using the small dip net, a fish is removed from the experimental chamber and transported to the necropsy area in the transport container.

• 1. The fish is placed in the euthanasia solution. The fish is removed from the solution when there is cessation of respiration and the fish is unresponsive to external stimuli.

• 1. The fish is wet weighed.

• 1. For preparation of tissues for VTG analysis, the fish can be placed on a corkboard on the stage of a dissecting microscope.
• (a) For zebrafish the head is cut right behind the pectoral fin and tail is cut right behind the dorsal fin.
• (b) For Japanese medaka the abdomen is opened via a carefully made incision that extends along the ventral midline from the pectoral girdle to a point just cranial to the anus. Using the small forceps and small scissors, the liver is carefully removed.

• 1. Specimen for VTG analysis are placed in eppendorf tubes and immediately frozen in liquid nitrogen.

• 1. The carcass including the gonads is placed into a pre-labelled plastic tissue cassette, which is transferred into Davidson's or Bouin's fixative. The volume of fixative should be at least 10 times the approximated volume of the tissues. The fixative container is gently agitated for five seconds to dislodge air bubbles from the cassette.

• 1. • (a) All tissues remain in Davidson's fixative overnight, followed by transfer to individual containers of 10 % neutral buffered formalin the next day. Containers with cassettes are gently agitated for 5 seconds to ensure adequate penetration of formalin into cassettes.
• (b) Tissues remain in Bouins fixative for 24 h, followed by transfer to 70 % ethanol.

Tissue Processing

Objectives:

• 1. Dehydrate tissue for adequate penetration of paraffin.

• 1. Impregnate the tissue with paraffin to maintain tissue integrity and create a firm surface for microtomy.

Procedures:

• 1. Labelled tissue cassettes are removed from formalin/ethanol storage and the cassettes are placed in the processing basket(s). The processing basket is loaded in the tissue processor.

• 1. The processing schedule is selected.

• 1. After the tissue processor has completed the processing cycle, the basket(s) may be transferred to the embedded station.

Embedding

Objective:

Properly orient the specimen in solidified paraffin for microtomy.

Procedures:

• 1. The basket(s) of cassettes is/are removed from the processor and immersed in the paraffin-filled front chamber of the embedding station thermal console or the cassettes are moved to a separate paraffin heater.

• 1. The first cassette to be embedded is removed from the front chamber of the thermal console or the paraffin heater. The cassette lid is removed and discarded, and the cassette label is checked against the animal records to resolve potential discrepancies prior to embedding.

• 1. An appropriately sized embedding mould is selected.

• 1. The mould is held under the spout of the dispensing console and filled with molten paraffin.

• 1. The specimen is removed from the cassette and placed in the molten paraffin in the mould. This is repeated with 4-8 specimens for each paraffin mould. The position of individual fish is marked by putting fish no 1 in 180 degrees to fish 2-4/8.

• 1. Additional paraffin is added to cover the specimen.

• 1. The mould with the cassette base is placed on the cooling plate of the cryo console.

• 1. After the paraffin has solidified, the block (i.e., the hardened paraffin containing the tissues and the cassette base) is removed from the mould.

Microtomy

Objective:

Cut and mount histological sections for staining.

Procedures:

• 1. The initial phase of microtomy termed ‘facing’ is conducted as follows:
• (a) The paraffin block is placed in the chuck of the microtome.
• (b) The chuck is advanced by rotating the microtome wheel and thick sections are cut from the paraffin surface of the block until the knife reaches the embedded tissues.
• (c) The section thickness on the microtome is set between 3 - 5 microns. The chuck is advanced and multiple sections are cut from the block to remove any artefacts created on the cut surface of the tissue during rough trimming.
• (d) The block can be removed from the chuck and placed facedown on ice to soak the tissue.

• 1. The next phase of microtomy is final sectioning and mounting of tissue sections on slides. These procedures are conducted as follows:
• (a) If the block has been placed on ice, the block is removed from the ice and replaced in the chuck of the microtome.
• (b) With the section thickness on the microtome set to 3 - 5 microns, the chuck is advanced by rotating the microtome wheel. Sections are cut from the block until a ‘ribbon’ containing at least one acceptable section including the gonads has been produced. (As necessary during sectioning, the block may be removed from the chuck, placed on ice to soak the tissue, and replaced in the chuck.)
• (c) The sections are floated flat on the surface of the water in the water bath. An attempt is made to obtain at least one section that contains no wrinkles and has no air bubbles trapped beneath it.
• (d) A microscope slide is immersed beneath the best section, which is lifted out of the water using the slide. This process is referred to as ‘mounting’ the section on the slide.
• (e) Three sections are prepared for a set of fish. The second and third sections are taken at 50 micron intervals following the first section. If the fish are not embedded with their gonads in the same sectioning level, more sections are to be made to ensure that at least six sections including the gonads are obtained from each fish.
• (f) With a slide-marking pen, the block number from which the slide was produced is recorded on the slide.
• (g) The slide is placed in a staining rack.
• (h) The block is removed from the chuck and placed facedown for storage.

Staining, Cover slipping, and Slide Labelling

Objectives:

• — Stain the sections for histopathological examination

• — Permanently seal mounted and stained tissues.

• — Permanently identify stained sections in a manner that allows complete traceability.

Procedures:

• 1. Staining
• (a) Slides are air-dried overnight before staining.
• (b) The sections are stained by Hematoxylin-Eosin.

• 1. Cover slipping
• (a) Cover slips can be applied manually or automatically.
• (b) A slide is dipped in xylene or TissueClear, and the excess xylene/TissueClear is gently knocked off the slide.
• (c) Approximately 0,1 ml of mounting medium is applied near the end of the slide opposite to the frosted end or on the cover slip.
• (d) The cover slip is tilted at a shallow angle as it is applied to the slide.

• 1. Labelling
• (a) Each slide label should contain the following information.
• (i) Laboratory name
• (ii) Species
• (iii) Specimen No./Slide No.
• (iv) Chemical/Treatment group
• (v) Date

Appendix 8

Statistical Flow Chart for vitellogenin analysis

Image omitted; see official source

Text of image

Tamhane-Dunnett test on nested ANOVA

Dunnett test on nested ANOVA

Dunnett test

variances stabilising transform?

Variances unequal

Variances equal

No

Yes

Yes

Step-down trend test on replicate means

<=3 reps per conc

variances stabilising transform?

Dunn or Mann-Whitney test

Normalising transform?

Nested ANOVA not normal

Nested ANOVA normal

Monotone

Determine whether Dose-Response is monotone

Drop water control

Dunn or Mann-Whitney test on rep means

Dunn test on rep means

=4 reps per conc

<=3 reps per conc

Dunn test

Tamhane-Dunnett test

=4 reps per conc

Step-down Jonckheere test

Step-down Jonckheere or Williams' test

Rep means not normal or not homogenous

Rep means normal & homogenous

Rep means not normally distributed

Rep means normally distributed

Not monotone

Combine controls, retains subgroups

Compare controls using Wilcoxon or T-test. Do controls differ?

Both solvent and non-solvent control are present

No

No

No

Yes

No

No

Yes

Yes

Yes

Statistical Flow Chart for sex ratio analysis

Image omitted; see official source

Text of image

Apply step-down Jonckheere-Terpstra test (+) to determine NOEC

Dunn or Mann-Whitney U-test w/Bonferroni-Holm adjustment to determine NOEC

Yes

Use Dunnett test if homogenous variances (*), Tamhane-Dunnett (T3) test otherwise, to determine NOEC

Are data normally distributed? (*)

Are data consistent with monotone dose-response?

(‡) Or other agreed control selection

(*) After arcsin square-root transform

(+) With fewer than 5 experimental units per treatment exact J-T or M-W test should be used if available

No

No

Yes

No

Combine controls

Drop water control (‡)

Compare controls using t-Test. Do controls differ?

No

Yes

Yes

Is solvent used?

Appendix 9

Guidance for tissue sampling for genetic sex determination and for genetic sex determination by PCR-method

Tissue sampling, preparation and storage before determination of genetic sex by PCR-method in medaka (Prepared by the Laboratory for Aquatic Organisms of Bayer CropScience AG)

• 1.With fine scissors the anal or the dorsal fin will be cut off in each individual fish and placed into a tube filled with 100 μl of extraction-buffer 1 (details on buffer preparation see below). The scissors will be cleaned after each single fish in a beaker filled up with distilled H2O and dried with a paper tissue.

• 2.Now the fin-tissues will be homogenised by a micro tube teflon pistil for the lysis of cells. For each tube a new pistil will be used to prevent any contaminations. The pistils will be placed overnight in 0,5 M NaOH, rinse for 5 minutes in distilled H2O and stored in ethanol or sterile after autoclave until use.

• 3.It is also possible to store the fin tissue without any extraction-buffer 1 on dry-ice and then at – 80 °C refrigerator to prevent any degeneration of the DNA. But the extraction runs better, if you extract the DNA at the same time (handling see above; samples should be thawed on ice after storaging at – 80 °C before the buffer will be filled in the tubes).

• 4.After homogenizing all tubes will be placed in a water bath and boiled for 15 minutes at 100 °C.

• 5.Then 100 μl of the extraction buffer 2 (details on buffer preparation see below) will be pipetted into each tube. The samples will be stored at room temperature for 15 minutes and in the meantime they will be sometimes gently shaken by hand.

• 6.Afterwards all tubes will be placed in the water bath again and boiled for another 15 minutes at 100 °C.

• 7.Until further analysis the tubes will be frozen at – 20 °C.

Buffer preparation

PCR-buffer 1:

500 mg N-Lauroylsarcosine (e.g. Merck KGaA, Darmstadt, GE)

2 ml 5M NaCl

ad 100 ml dest. H2O

→ autoclave

PCR-buffer 2:

20 g Chelex (e.g. Biorad, Munich, GE)

To swell in 100 ml dest. H2O

→ autoclave

Determination of genetic sex (by PCR-method) in medaka (Prepared by the Laboratory for Aquatic Organisms of Bayer CropScience AG and Universität Würzburg Biozentrum)

The prepared and frozen tubes (described in the above section) will be thawed on ice. After that, they will be centrifuged using an Eppendorf centrifuge (30 sec at max. speed, at room temperature). For the PCR, the clear supernatant separated from the precipitate will be used. It has absolutely to be avoided that any traces of Chelex (localized in the precipitate) are transferred to the PCR reaction, because this will interfere with the ‘Taq’-polymerase activity. The supernatant will be used directly or can be stored frozen (at – 20 °C) and rethawed again in several cycles without negative impact on the DNA for later analyses.

1. Preparation of the ‘Reaction Mix’ (25 μl per sample):

	Volume	Final Concentration
Template DNA	0,5μl-2μl
10xPCR-buffer with MgCl2	2,5μl	1x
Nucleotides (each of dATP, dCTP, dGTP, dTTP)	4μl (5mM)	200μM
Forward Primer (10μM) (see below 3-5)	0,5μl	200nM
Reverse Primer (10μM) (see below 3-5)	0,5μl	200nM
DMSO	1,25μl	5 %
Water (PCR grade)	up to 25μl
Taq E- Polymerase	0,3μl	1,5U
10xPCR-buffer with MgCl2: 670mM Tris/HCl (pH8,8 at 25 °C), 160mM (NH4)2SO4, 25mM MgCl2, 0,1 %Tween 20	10xPCR-buffer with MgCl2: 670mM Tris/HCl (pH8,8 at 25 °C), 160mM (NH4)2SO4, 25mM MgCl2, 0,1 %Tween 20	10xPCR-buffer with MgCl2: 670mM Tris/HCl (pH8,8 at 25 °C), 160mM (NH4)2SO4, 25mM MgCl2, 0,1 %Tween 20

For each PCR (see below 3-5) the special primer as a new combination of ‘Reaction-Mix’ and the adequate needed amount of template DNA for each sample (see above) is needed. The respective volumes will be transferred into new tubes using pipettes. After that all tubes will be closed, stirred (ca. 10 sec) and centrifuged (10 sec, at room temperature). Now the respective PCR-programmes can be started. Additionally a positive control (exemplary DNA sample with known activity and clear results) and a negative control (1 μl dest. H2O) will be used in each PCR-programme.

2. Preparation of the agarose gel (1 %) — During running PCR-programmes:

• — Solve 3 g agarose in 300 ml 1 × TAE-buffer (1 % agarose gel)

• — This solution should be boiled using an microwave (ca. 2-3 min)

• — Transfer the hot solution into a special casting box, which lies on ice

• — After ca. 20 min the agarose gel is ready to use

• — Storage the agarose gel in 1 × TAE-buffer until the end of the PCR-programmes

3. Actin-PCR-programme:

This PCR-reaction is aimed to demonstrate that the DNA in the sample is not harmed.

• — Special primer: ‘Mact1(upper/forward)’ → TTC AAC AGC CCT GCC ATG TA ‘Mact2(lower/reverse)’ → GCA GCT CAT AGC TCT TCT CCA GGG AG

• — Programme: 5 min 95 °C Cycle (35-times): Denaturation → 45 sec at 95 °C Annealing → 45 sec at 56 °C Elongation → 1 min at 68 °C 15 min 68 °C

4. X- and Y-Gene-PCR-programme:

The samples with intact DNA will be used in this PCR-programme to detect the X- and Y-Genes. Male DNA should show one double-band and female DNA should show one single band (after staining and gel-electrophoresis). For this programme-run one positive control for males (XY-sample) and one for females (XX-sample) should be included.

• — Special primer: ‘PG 17,5’ (upper/forward) → CCG GGT GCC CAA GTG CTC CCG CTG ‘PG 17,6’ (lower/reverse) → GAT CGT CCC TCC ACA GAG AAG AGA

• — Programme: 5 min 95 °C Cycle (40-times): Denaturation → 45 sec at 95 °C Annealing → 45 sec at 55 °C Elongation → 1 min 30 sec at 68 °C 15 min 68 °C

5. Y-Gene-PCR-programme as ‘control’ for X- and Y-Gene-PCR-programme:

This PCR-programme verifies the results of the ‘X- and Y-Gene-PCR-programme’. The ‘male-samples’ should show one band and the ‘female-samples’ shouldn't show any band (after staining and gel-electrophoresis).

• — Special primer: ‘DMTYa (upper/forward)’ → GGC CGG GTC CCC GGG TG ‘DMTYd (lower/reverse)’ → TTT GGG TGA ACT CAC ATG G

• — Programme: 5 min 95 °C Cycle (40-times): Denaturation → 45 sec at 95 °C Annealing → 45 sec at 56 °C Elongation → 1 min at 68 °C 15 min 68 °C

6. Staining of the PCR-samples:

Staining solution:

50 % Glycerol

100 mM EDTA

1 % SDS

0,25 % Bromphenolblue

0,25 % Xylenecyanol

Pipette 1 μl of the staining solution into each single tube

7. Start of the Gel-Electrophoresis:

• — The prepared 1 % agarose gel will be transferred into a gel-electrophoresis-chamber filled with 1 × TAE-Buffer

• — 10 - 15 μl of each stained PCR-sample will be pipetted into an agarose gel slot

• — Also 5 - 15 μl of the 1kb-‘Ladder’(Invitrogen) will be pipetted into a separate slot

• — Start the electrophoresis by 200 V

• — Stop after 30-45 min

8. Determination of the bands:

• — Clean the agarose gel in distilled H2O

• — Now transfer the agarose gel into Ethidium bromide for 15 - 30 min

• — After that, a picture of the agarose gel should be taken in an UV-light-box

• — Finally the samples are analysed in comparison to the positive control-band (or bands) and the ladder

Appendix 10

Guidance on tissue sampling for genetic sex determination by PCR method in the three-spined stickleback

Tissue sampling and DNA extraction

DNA can be extracted using a variety of commercially available reagents and both manual and automated extraction systems. The protocol used at the Cefas Weymouth laboratory is outlined below, and the alternative approaches have been added where appropriate.

• 1.With fine scissors, a small piece of tissue (10-20 mg) from the dorsolateral area (after removing the head and tail for VTG analysis), is removed from each individual fish. The tissue is added into a tube and either placed directly in liquid nitrogen (for storage at – 80 °C) or filled with 70 % ethanol (for transport and subsequent storage at 4 °C). The scissors are cleaned after each single fish in 70 % ethanol then in distilled water and dried with tissue paper.

• 2.The ethanol (if present) is removed by aspiration and the tissue is digested overnight with proteinase K in 400 μl of ATL buffer (Qiagen). An aliquot (200 μl) of the digest is transferred to a 96-well S-block (Qiagen) and the DNA extracted in a 96-well format using the Qiagen Universal BioRobot and the QIamp Investigator BioRobot kit. The DNA is eluted in a 50 μl of DNase and RNase free water. If using hard tissues to extract DNA (such as a spine or a pectoral fin) it may be necessary to homogenise the sample in the lysis buffer using a FastPrep® tissue lyser or equivalent tissue disruption system.

• Alternatively,

• (a) the tissue is digested overnight with proteinase K in 400 μl of G2 lysis buffer (Qiagen) and DNA is extracted from 200 μl of the digest using either the EZ-1 DNA easy tissue kit and the EZ-1 biorobot or the DNA easy tissue mini kit. The DNA is eluted in a 50 μl volume.

• (b) The tissues are processed using the DNAzol reagent. Briefly, tissue samples are lysed in 1ml of DNAzol for 10 minutes in a 1,5 ml micro centrifuge tube and then centrifuged at 13 000 rpm for 5 minutes to remove any particulate matter. The lysed sample is then transfered to a new 1,5 ml micro centrifuge tube containing 500 μl of 100 % molecular grade ethanol and then centrifuged at 13 000 rpm for 10 minutes to precipitate the DNA. The ethanol is removed and replaced with 400 μl of 70 % molecular grade ethanol, centrifuged at 13 000 rpm for 5 minutes and the DNA pellet is dissolved in 50 μl molecular DNase and RNase free water. Again, when using the hard tissues (pectoral fin) it may be necessary to homogenise the sample in the lysis buffer using a FastPrep® tissue lyser or equivalent tissue disruption system prior to extracting the DNA.

• 3.The DNA is stored at – 20 °C until required.

Important note: gloves must be worn during the procedures.

Polymerase chain reaction (PCR) analysis

Amplifications were performed using 2,5 μl of the DNA extract in a 50 μl reaction volume using the Idh locus primers (as described by Peichel et al., 2004. Current Biology 1:1416-1424):

Forward primer	5' GGG ACG AGC AAG ATT TAT TGG 3'
Reverse primer	5' TAT AGT TAG CCA GGA GAT GG 3'

There are numerous suppliers of suitable PCR reagents. The method outlined below is that currently used at the Cefas Weymouth laboratory.

1. Preparation of the ‘Reaction Mix’ (50 μl per sample):

A mastermix is prepared as follows. This can be prepared in advance and stored frozen at – 20 °C until required. Make sufficient mastermix for a negative control (molecular biology grade water only).

	Volume (stock conc.)/ sample	Final Concentration
5xGoTaq® Reaction Buffer	10μl	1x
MgCl2	5 μl (25 mM)	2,5 mM
Nucleotides (dATP, dCTP, dGTP, dTTP)	0,5 μl (25 mM each)	250 μM each
Forward Primer	0,5μl (0,1 nmol/μl)	2,0 μM
Reverse Primer	0,5μl (0,1 nmol/μl)	2,0μM
Molecular biology grade water	30,75 μl
GoTaq polymerase	0,25 μl	1,25U

• — Dispense 47,5 μl to a labelled 0,5 ml thin walled PCR tube.

• — Add 2,5 μl of the purified DNA to the appropriately labelled tube. Repeat for all samples and the negative control.

• — Over lay with 2 drops of mineral oil. Alternatively, use a thermal cycler with a heated lid.

• — Close the lids.

• — Samples were denatured in a Peltier PTC-225 thermal cycler at 94 ± 2 °C for 5 minutes followed by 39 cycles of 94 ± 2 °C for 1 minute, 55 ± 2 °C for 1 minute, 72 ± 2 °C for 1 minute, and a final extension of 72 ± 2 °C for 10 minutes.

2. Preparation of the agarose gel (2 %):

Traditionally the PCR products are resolved on a 20 % agarose gel containing ethidium bromide.

Capillary based electrophoresis systems can also be used.

• — Weigh 2 g agarose in 100 ml 1 × TAE-buffer

• — Heat in a microwave (ca. 2-3 min) to dissolve the agarose.

• — Add 2 drops of ethidium bromide final concentration 0,5 μg/ml

• — Transfer the hot solution into the gel casting equipment.

• — Allow the gel to harden

3. Gel-Electrophoresis:

• — Transferred the agarose gel to the electrophoresis equipment and submerge in 1 × TAE-buffer

• — Load 20 μl of each sample to a separate well, adding a molecular weight marker (100 bp DNA ladder, Promega) to a spare well.

• — Electrophoresis is performed at 120 V for 30-45 minutes.

4. Visualisation of the amplification products

If the ethidium bromide was incorporated in to the agarose gel as described above, the DNA products are visualised under a UV source. Alternatively the agarose gel is stained by covering the gel in a dilute solution of ethidium bromide (0,5 μg/ml in water) for 30 minutes prior to visualisation.

Appendix 11

Guidance for artificial fertilisation procedure for the three-spined stickleback

The purpose of this section is to describe the procedures to obtain fertilised eggs from the three-spined stickleback in view of using them in the FSDT.

Procedures

Obtaining sperm from the males

• 1.A well-coloured male of the desired population is euthanised.

• 2.The testes are dissected from each side of the fish. The testes are generally heavily pigmented, rod shaped structures that are readily apparent at the lateral midline of the body. Use either of the following methods:

• 3.Using a pair of fine scissors, begin at the cloaca and make a 1-1,5 cm incision with a single snip angled at about 45 degrees.

• 4.Use a scalpel to make a small incision in the side of the fish slightly posterior to the pelvis and just ventral of the lateral plates.

• 5.The testes are removed using fine forceps and placed into a petri dish.

• 6.Each testis is covered with 100 μl freshly made Hank's final solution ([^E0209]).

• 7.The testes are finely diced by using a razor blade or scalpel. This will release sperm and give the Hank's solution a milky appearance.

• 8.The fluid containing sperm is added into a tube, while trying not to include any pieces of testes tissue when pipetting.

• 9.800 μl of Hank's final solution are added into the tube and mixed well.

• 10.If required, the male can be preserved by fixing in 100 % ethanol or other desired fixative. This is particularly important if the study is assigning parental origin of offsprings.

Important note: Although most of the stock solutions required can be made in advance, stock 5 and subsequently the final solution, should be made up fresh on the day of use.

Stock 1	Stock 1
NaCl	8,00 g
KCl	0,40 g
Distilled water (DW)	100 ml
Stock 2	Stock 2
Na2HPO4 (anhydrous)	0,358 g
KH2PO4	0,60 g
DW	100 ml
Stock 3	Stock 3
CaCl2	0,72 g
DW	50 ml
Stock 4	Stock 4
MgSO4 .7H2O	1,23 g
DW	50 ml
Stock 5 (freshly prepared)	Stock 5 (freshly prepared)
NaHCO3	0,35 g
DW	10 ml

Note: If you already have some of the above salts but with different water content (i.e. 2H2O instead of anhydrous) you can still use it but first adjust weight based on molecular weight).

For Hank's final solution combine in the following order:

stock 1	1,0 ml
stock 2	0,1 ml
stock 3	0,1 ml
DW	8,6 ml
stock 4	0,1 ml
stock 5	0,1 ml

Mix well before use.

Fertilisation

• 1.Large, gravid females are identified from the desired population; females are ready for squeezing only when you can see eggs protruding from the cloaca. Ready females have the characteristic ‘head up’ posture.

• 2.Gently run a finger or thumb down the side of the fish towards the tail to encourage the expulsion of an egg sack into a fresh petri dish. Repeat on the other side and return the fish to its tank.

• 3.The eggs can be spread out (forming a monolayer) using a fine paintbrush. It is important to try and expose as many eggs as possible to the sperm so maximising the surface area of the eggs is helpful. Important note: Keep the eggs humid by laying damp tissue around them (it is important the eggs do not touch water directly as this can prematurely harden the chorion preventing fertilisation). There is a large variation in the number of eggs each female can produce but as an average, about 150 eggs should be easily obtained from a single gravid female.

• 4.25μl of sperm in Hank's mixture is spread evenly over the whole surface of the eggs using the paintbrush. The eggs will quickly harden and change colour (within a minute) once fertilisation has begun. If the estimated number of eggs is more than 150, repeat the procedure. Similarly if the eggs don't harden within a minute add a bit more sperm. Important note: Adding more sperm does not necessarily improve fertilisation rate.

• 5.The eggs and the sperm solution should be left to ‘interact’ for at least 15 minutes and the fertilised eggs should be placed into the exposure aquaria within 1,5 hours post fertilisation.

• 6.The procedure is repeated using another female until the desired number of eggs is collected.

• 7.Spare few eggs from the last batch and fix them in 10 % acetic acid.

Counting and distributing eggs in test aquaria

• 1.Eggs should be evenly distributed between each treatment level to avoid genetic bias. Each batch of fertilised eggs should be separated into equal size groups (as many as the treatment levels) by the use of a blunt instrument (i.e. wide-blade entomology forceps or use of an inoculation loop). If you aim for 4 replicates per treatment, with 20 eggs each then you need to distribute 80 eggs per exposure aquaria. Important note: It is advisable to add an extra 20 % (i.e. 96 eggs per treatment level) until you are confident that you obtain 100 % fertilisation rates.

• 2.Stickleback eggs are very prone to fungal infections outside the father-guarded nest. In this respect, treatment of all eggs with methylene blue during the first 5 days of the test is critically important. A stock solution of methylene blue is prepared at 1 mg/ml and added to the exposure aquaria to give a maximum final concentration of 2,125 mg/l. Important note: Sticklebacks should not be exposed to methylene blue once hatched so the system should be free of methylene blue by day 6.

• 3.The eggs are inspected daily and any dead or unfertilised eggs are recorded as such. Important note: The eggs should never be outside water until they hatch even for very brief periods.

C.42.   BIODEGRADABILITY IN SEAWATER

GENERAL INTRODUCTION

• 1.This test method is equivalent to OECD Test Guideline (TG) 306 (1992). When the original test methods were developed, it was not known to what extent results from the screening tests for ready biodegradability using freshwater, and sewage effluent or activated sludge as inoculum, could be applied to the marine environment. Variable results on this point have been reported (e.g. (1)).

• 2.Many industrial waste waters, containing a variety of chemicals, reach the sea either by direct discharge or via estuaries and rivers in which the residence times are low compared with the period necessary for complete biodegradation of many of the chemicals present. Because of the growing awareness of the need to protect the marine environment against increasing loads of chemicals and the need to estimate the probable concentration of chemicals in the sea, test methods for biodegradability in seawater have been developed.

• 3.The methods described here use natural seawater both as the aqueous phase and as the source of micro-organisms. In an endeavour to conform with the methods for ready biodegradability in freshwater, the use of ultra-filtered and centrifuged seawater was investigated, as was the use of marine sediments as inocula. These investigations were unsuccessful. The test medium therefore is natural seawater pre-treated to remove coarse particles.

• 4.In order to assess ultimate biodegradability with the Shake Flask Method, relatively high concentrations of the test substance have to be used because of the poor sensitivity of the dissolved organic carbon (DOC) analytical method. This in turn necessitates the addition to the seawater of mineral nutrients (N and P), the low concentrations of which would otherwise limit the removal of DOC. It is also necessary to add the nutrients in the Closed Bottle Method because of the concentration of the added test substance.

• 5.Hence, the methods are not tests for ready biodegradability since no inoculum is added in addition to the micro-organisms already present in the seawater. Neither do the tests simulate the marine environment since nutrients are added and the concentration of test substance is very much higher than would be present in the sea. For these reasons the methods are proposed under a new subsection ‘Biodegradability in Seawater’.

APPLICATION

• 6.The results of the tests, which would be applied because the pattern of use and disposal of the substance in question indicated a route to the sea, give a first impression of biodegradability in seawater. If the result is positive (> 70 % DOC removal; > 60 % ThOD — theoretical oxygen demand), it may be concluded that there is a potential for biodegradation in the marine environment. However, a negative result does not preclude such a potential but indicates that further study is necessary, for example, using as low a concentration of the test substance as possible.

• 7.In either case, if a more definitive value for the rate or degree of biodegradation in seawater at a particular site is required, other more complex and sophisticated, and hence more costly, methods would have to be applied. For example, a simulation test could be applied using a concentration of test substance nearer to the likely environmental concentration. Also, non-fortified, non-pre-treated seawater taken from the location of interest could be used and primary biodegradation could be followed by specific chemical analysis. For ultimate biodegradability, ¹⁴C-labelled substances would be necessary in order that the rates of the disappearance of soluble organic ¹⁴C and the production of ¹⁴CO2 at environmentally realistic concentrations could be measured.

CHOICE OF METHODS

• 8.The selection of which method to use depends on a number of factors; the following Table is given to help the selection. While substances of water solubility below the equivalent of about 5 mg C/l cannot be tested in the Shake Flask Method, at least, in principle, poorly soluble substances may be tested in the Closed Bottle Method.

Table

Advantages and disadvantages of the shake flask and closed bottle test

METHOD	ADVANTAGES	DISADVANTAGES
SHAKE FLASK	— simple apparatus except C analyser — 60 d duration is not a problem — no interference from nitrification — can be adapted for volatile substances	— needs C analyser — uses 5-40 mg DOC/1, could be inhibitory — DOC determination is difficult at low concentrations in seawater (chloride effect) — DOC sometimes high in seawater
CLOSED BOTTLE	— simple apparatus — simple end determination — uses low concentration of test substance (2 mg/l) thus less chance of inhibition — easily adapted for volatile substances	— could be difficult to maintain air-tightness of bottles — wall growth of bacteria can lead to false values — blank O2 uptake values can be high especially after 28 days; could be overcome by ageing the seawater — possible interference from O2 uptake by nitrification

SHAKE FLASK METHOD

INTRODUCTION

• 1.This method is a seawater variant of the Modified OECD Screening Test described in Chapter C.4B of this Annex (2). It was finalised as a result of a ring test organized for the European Commission (EC) by the Danish Water Quality Institute (3).

• 2.In common with the accompanying marine Closed Bottle Method, the results from this test are not to be taken as indicators of ready biodegradability, but are to be used specifically for obtaining information about the biodegadability of substances in marine environments.

PRINCIPLE OF THE METHOD

• 3.A pre-determined amount of the test substance is dissolved in the test medium to yield a concentration of 5-40 mg dissolved organic carbon (DOC)/l. If the limits of sensitivity of organic carbon analyses are improved, the use of lower concentrations of test substance may be advantageous, particularly for inhibitory substances. The solution of the test substance in the test medium is incubated under agitation in the dark or in diffuse light under aerobic conditions at a fixed temperature (controlled to ± 2 °C) which will normally be within the range 15-20 °C. In cases where the objective of the study is to simulate environmental situations, tests may be carried out beyond this normal temperature range. The recommended maximum test duration is about 60 days. Degradation is followed by DOC measurements (ultimate degradation) and, in some cases, by specific analysis (primary degradation).

INFORMATION ON THE TEST SUBSTANCE

• 4.In order to know whether the test may be applied to a particular substance, some of its properties must be known. The organic carbon content of the substance must be established, its volatility must be such that significant losses do not occur during the course of the test and its solubility in water should be greater than the equivalent of 25-40 mg C/l. Also, the test substance should not significantly adsorb onto glass surfaces. Information on the purity or the relative proportions of major components of the test substance is required in order that the results obtained can be interpreted, especially when the result lies close to the ‘pass’ level.

• 5.Information on the toxicity of the test substance to bacteria, for example as measured in short-term respiration rate tests (4), may be useful when selecting appropriate test concentrations and may be essential for the correct interpretation of low biodegradation values. However, such information is not always sufficient for interpreting results obtained in the biodegradation test and the procedure described in paragraph 18 is more suitable.

REFERENCE SUBSTANCES

• 6.Suitable reference substances must be used to check the microbial activity of the seawater sample. Sodium benzoate, sodium acetate and aniline are examples of substances which may be used for this purpose. The reference substances must be degraded within a reasonably short time span, otherwise it is recommended that the test be repeated using another seawater sample.

• 7.In the EC ring test where seawater samples were taken at different locations and at different times of the year (3), the lag phase (tL) and time to achieve 50 per cent degradation (t50), excluding the lag phase, were 1 to 4 days and 1 to 7 days respectively for sodium benzoate. For aniline the tL ranged from 0 to 10 days, whilst the t50 ranged from 1 to 10 days.

REPRODUCIBILITY AND SENSITIVITY OF THE METHOD

• 8.The reproducibility of the method was established in the ring test (3). The lowest concentration of test substance, for which this method can be used with DOC analysis, is largely determined by the detection limit of the organic carbon analysis (about 0,5 mg C/l, at present) and the concentration of dissolved organic carbon in the seawater used (usually of the order of 3-5 mg/l for water from the open sea). The background concentration of DOC should not exceed about 20 % of the total DOC concentration after addition of test substance. If this is not feasible, the background concentration of DOC may sometimes be reduced by ageing the seawater prior to testing. If the method is used with specific chemical analysis only (by which primary degradation is measured), the investigator must document, by supplying additional information, whether ultimate degradability can be expected. This additional information may consist of the results from other tests for ready or inherent biodegradability.

DESCRIPTION OF THE METHOD

Apparatus

• 9.Normal laboratory apparatus and:

• a. Shaking machine accommodating 0,5-2 litre Erlenmeyer flasks, either with automatic temperature control or used in a constant temperature room at 15-20 °C controlled to ± 2 °C;

• b. Narrow neck, 0,5-2 litre Erlenmeyer flasks;

• c. Membrane filtration apparatus, or centrifuge;

• d. Membrane filters, 0,2-0,45 μm;

• e. Carbon analyser;

• f. Equipment for specific analysis (optional).

Seawater

• 10.Collect a sample of seawater in a thoroughly cleansed container and transport to the laboratory, preferably within one or two days of collection. During transport, do not allow the temperature of the sample to exceed significantly the temperature to be used in the test. Identify the sampling location precisely and describe it in terms of its pollutional and nutrient status. Especially for coastal waters, include in this characterization a heterotrophic microbial colony count and the determination of the concentrations of dissolved nitrate, ammonium and phosphate.

• 11.Provide the following information for the seawater sample itself:

• — date of collection;

• — depth of collection;

• — appearance of sample — turbid, etc.;

• — temperature at the time of collection;

• — salinity;

• — DOC;

• — delay between collection and use in the test.

• 12.If the DOC content of the seawater sample is found to be high (paragraph 8), it is recommended that the seawater be aged for about a week prior to use. Age by storing under aerobic conditions at the test temperature and in the dark or in diffuse light. If necessary, maintain aerobic conditions by gentle aeration. During ageing, the content of easily degradable organic material is reduced. In the ring test (3), no difference was revealed between the degradation potential of aged and freshly collected seawater samples. Prior to use, pre-treat the seawater to remove coarse particles, e.g. by filtration through a nylon filter or coarse paper filter (not membrane or GF-C filters), or by sedimentation and decanting. The procedure used must be reported. Carry out pre-treatment after ageing, if used.

Stock solutions for mineral nutrients

• 13.Prepare the following stock solutions, using analytical grade reagents:

(a)	Potassium dihydrogen orthophosphate, KH2PO4	8,50 g
(a)	Dipotassium hydrogen orthophosphate, K2HPO4	21,75 g
(a)	Disodium hydrogen orthophosphate dihydrate, Na2HPO4·2H2O	33,30 g
(a)	Ammonium chloride, NH4Cl	0,50 g
(a)	Dissolve and make up to 1 litre with distilled water.
(b)	Calcium chloride, CaCl2	27,50 g
(b)	Dissolve and make up to 1 litre with distilled water.
(c)	Magnesium sulphate heptahydrate, MgSO4·7H2O	22,50 g
(c)	Dissolve and make up to 1 litre with distilled water.
(d)	Iron (III) chloride hexahydrate, FeCl3·6H2O	0,25 g
(d)	Dissolve and make up to 1 litre with distilled water.

• Precipitation in solution (d) may be prevented by adding one drop of concentrated HCl or 0,4 g ethylenediaminetetra-acetic acid (EDTA, disodium salt) per litre. If a precipitate forms in a stock solution, replace it with freshly made solution.

Preparation of test medium

• 14.Add 1 ml of each of the above stock solutions per litre of pre-treated seawater.

Inoculum

• 15.Do not add a specific inoculum in addition to the micro-organisms already present in the seawater. Determine (optionally) the number of colony-forming heterotrophs in the seawater test medium (and preferably also in the original seawater samples) e.g. by plate count, using marine agar. This is particularly desirable for samples from coastal or polluted sites. Check the heterotrophic microbial activity in the seawater by performing a test with a reference substance.

Preparation of flasks

• 16.Ensure that all glassware is scrupulously clean, not necessarily sterile, (e.g. using alcoholic hydrochloric acid), rinsed and dried before use in order to avoid contamination with residues from previous tests. The flasks must also be cleaned before first use.

• 17.Evaluate test substances in duplicate flasks simultaneously, together with a single flask for the reference substance. Carry out a blank test, in duplicate, with neither test nor reference substance for the determination of analytical blanks. Dissolve the test substances in the test medium — they may be conveniently added via a concentrated stock solution — to give the desired starting concentrations of normally 5-40 mg DOC/l. Test the reference substance normally at a starting concentration corresponding to 20 mg DOC/l. If stock solutions of test and/or reference substances are used, ensure that the salinity of the seawater medium is not greatly altered.

• 18.If toxic effects can be expected or cannot be ruled out, it may be advisable to include an inhibition experiment, in duplicate, in the test design. Add the test and reference substances to the same vessel, the concentration of the reference substance being normally the same as in the control test (i.e. 20 mg DOC/l) in order to allow comparison.

• 19.Dispense adequate amounts of test solutions into the Erlenmeyer flasks (up to about half the flask volume is a convenient amount) and subsequently provide each flask with a loose cover (e.g. aluminium foil) that makes gas exchange between the flask and the surrounding air possible. (Cotton wool plugs are unsuitable if DOC analysis is used). Place the vessels on the shaker and shake continuously at a gentle rate (e.g. 100 rpm) throughout the test. Control the temperature (15-20 °C and within ± 2 °C), and shield the vessels from light in order to avoid growth of algae. Ensure that the air is free of toxic materials.

Physical-chemical control test (optional)

• 20.If abiotic degradation or loss mechanisms are suspected, such as hydrolysis (a problem with specific analysis only), volatilization, or adsorption, it is advisable to perform a physical-chemical control experiment. This can be done by adding mercury (II) chloride (HgCl2) ([^E0210]) (50-100 mg/l) to vessels with test substance in order to stop microbial activity. A significant decrease in DOC or specific substance concentration in the physical-chemical control test indicates abiotic removal mechanisms. (If mercury chloride is used, attention should be paid to interferences or catalyst poisoning in DOC analysis.)

Number of flasks

• 21.In a typical run, the following flasks are used:

Flasks 1 & 2	—	containing test substance (test suspension);
---	---	---
Flasks 3 & 4	—	containing seawater only (blank);
---	---	---
Flask 5	—	containing reference substance (procedure control);
---	---	---
Flask 6	—	containing test and reference subtance (toxicity control) — optional;
---	---	---
Flask 7	—	containing test substance and sterilising agent (abiotic sterile control)-optional.

DOC analysis

• 22.In the course of the test, withdraw samples at suitable intervals for DOC analysis (Appendix 1). Always take samples at the start of the test (day 0) and at day 60. A minimum of five samples in total are required to describe the time-course of degradation. No fixed time schedule for sampling can be stated as the rate of biodegradation varies. Carry out the DOC determination in duplicate on each sample.

Sampling

• 23.The required volume of the samples depends upon the analytical method (specific analysis), on the carbon analyser used, and on the procedure (membrane filtration or centrifugation) selected for sample treatment before carbon determination (paragraphs 25 and 26). Before sampling ensure that the test medium is mixed well and that any material adhering to the wall of the flask is dissolved or suspended.

• 24.Membrane-filter or centrifuge immediately after sampling. If necessary, store the filtered or centrifuged samples at 2-4 °C for up to 48 hours or below – 18 °C for longer periods (if it is known that the substance will remain unaffected, acidify to pH 2 before storing).

• 25.Membrane filters (0,2-0,45 μm) are suitable if it is ensured that they neither release carbon nor adsorb the substance in the filtration step e.g. polycarbonate membrane filters. Some membrane filters are impregnated with surfactants for hydrophilization and may release considerable quantities of dissolved carbon. Prepare such filters by boiling in deionised water for three consecutive periods, each of one hour. After boiling, store the filters in deionised water. Discard the first 20 ml of the filtrate.

• 26.Centrifugation of the samples may be chosen as an alternative to membrane filtration. Centrifuge at 40 000 m·s^{– 2} (~ 4 000 g) for 15 minutes, preferably in a refrigerated centrifuge.

Note: The differentiation of Total Organic Carbon (TOC) over DOC (TOC/DOC) by centrifugation at very low concentrations does not seem to work, since either not all bacteria are removed, or carbon as part of the bacterial plasma is redissolved. At higher test concentrations (> 10 mg C per litre), the centrifugation error seems to be comparatively small.

Frequency of sampling

• 27.If analyses are performed immediately after sampling, assess the next sampling time by considering the result of the analytical determination.

• 28.If samples are preserved (paragraph 24) for analysis at a later time, take more samples than the required minimum number of five. Analyse the last samples first, and by a step-wise ‘backwards’ selection of appropriate samples for analysis, it is possible to obtain a good description of the biodegradation curve with a relatively small number of analytical determinations. If no degradation has taken place by the end of the test, no further samples need to be analysed, and in this situation, the ‘backwards’ stategy may save considerable analytical costs.

• 29.If a plateau on the degradation curve is observed before the 60th day, end the test. If degradation has obviously started by day 60, but has not reached a plateau, extend the experiment for a further period.

DATA AND REPORTING

Treatment of results

• 30.Record the analytical results on the attached data sheet (Appendix 2), and calculate the biodegradation values for both test and reference substances from the equation:

Image omitted; see official source

• where:

Dt	=	degradation in percentage DOC or specific substance removal at time t,
---	---	---
Co	=	starting concentration of DOC or specific substance in the test medium,
---	---	---
Ct	=	concentration of DOC or specific substance in the test medium at time t,
---	---	---
Cbl(0)	=	starting concentration of DOC or specific substance in the blank,
---	---	---
Cbl(t)	=	concentration of DOC or specific substance in the blank at time t.

• 31.State degradation as the percentage DOC removal (ultimate degradation) or specific substance removal (primary degradation) at time t. Calculate the DOC concentrations to the nearest 0,1 mg per litre, and round up the means of the Dt values to the nearest whole per cent.

• 32.Illustrate the course of the degradation graphically in a diagram as shown in the figure in ‘Validity and interpretation of results’. If there are sufficient data, calculate from the curve the lag phase (tL) and the time to reach 50 per cent removal from the end of the lag phase (t50).

Test report

• 33.The test report must contain the following information:

Test substance:

• — physical nature and, where relevant, physicochemical properties;

• — identification data.

Test conditions:

• — location and description of the sampling site; pollutional and nutrient status (colony count, nitrate, ammonium, phosphate if appropriate);

• — characteristics of the sample (date of sampling, depth, appearance, temperature, salinity, DOC (optional), delay between collection and use in the test;

• — method used (if any) for ageing of the seawater;

• — method used for pre-treatment (filtration/sedimentation) of the seawater;

• — method used for DOC determination;

• — method used for specific analysis (optional);

• — method used for determining the number of heterotrophs in the seawater (plate count method or alternative procedure) (optional);

• — other methods (optional) used to characterise the seawater (ATP measurements, etc.).

Results:

• — analytical data reported on a data sheet (Appendix 2);

• — the course of the degradation test is represented graphically in a diagram showing the lag phase (tL), slope, and time (starting from the end of the lag phase) to reach 50 per cent removal (t50). The lag phase may be estimated graphically as shown in the figure in the ‘Validity and interpretation of results’ section or conveniently taken as the time needed for 10 per cent degradation;

• — percentage degradation measured after 60 days, or at end of test.

Discussion of results.

Validity and interpretation of results

• 34.The results obtained with the reference substances e.g. sodium benzoate, sodium acetate or aniline, should be comparable to results obtained in the ring test (3) (refer to section on ‘Reference substances’, paragraph 7). If results obtained with reference substances are atypical, the test should be repeated using another seawater sample. Although results of inhibition tests may not always be straightforward to interpret because of the contribution of DOC by the test substance, a significant reduction of the total DOC removal rate, compared with that of the control, is a positive sign of toxic effects.

• 35.Owing to the relatively high test concentrations used as compared with most natural systems (and consequently an unfavourable ratio between the concentrations of test substances and other carbon sources), the method is to be regarded as a preliminary test which can be used to indicate whether or not a substance is easily biodegradable. Accordingly a low result does not necessarily mean that the test substance is not biodegradable in marine environments, but indicates that more work will be necessary in order for this to be established.

• An example of a theoretical degradation experiment illustrating a feasible way of estimating the values of tL (length of ‘lag phase’) and t50 (time interval, starting at tL), needed to reach 50 per cent removal, is given in the figure below.

Image omitted; see official source

Text of image

Dunnett's Test

William Test

Transform data

Yes

Yes

Yes

Yes

No

Exclude control without solvent

Are both controls equal? U-Test

Are both controls equal? t-Test

Bonferroni — U-Test

Jonckheere-Terpstra Test

Shirley Test

Dunn's Test

Both controls might be pooled

Additional solvent control?

Additional solvent control?

Statistical testing not recommended

At least four replicates per treatment?

No success

Data:

Variation homogenous?

Distribution normal?

Yes

Yes

Yes

No

No

No

No

No

Exclude control without solvent

Both controls might be pooled

Start

Non-parametric Tests

Parametric Tests

CLOSED BOTTLE METHOD

INTRODUCTION

• 1.This method is a seawater variant of the Closed Bottle Test (5) and was finalised as a result of a ring test organised for the European Commission (EC) by the Danish Water Quality Institute (3).

• 2.In common with the accompanying marine Shake Flask Method, results of this test are not to be taken as indications of ready biodegradability, but are to be used specifically for obtaining information about the biodegradability of substances in marine environments.

PRINCIPLE OF THE METHOD

• 3.A pre-determined amount of the test substance is dissolved in the test medium in a concentration of usually 2-10 mg of test substance per litre (one or more concentrations may be used). The solution is kept in a filled closed bottle in the dark in a constant temperature bath or enclosure controlled to ± 1 °C within a range of 15-20 °C. In those cases where the objective of the study is to simulate environmental situations, tests may be carried out beyond this normal temperature range providing suitable adjustments are made for temperature control. The degradation is followed by oxygen analyses over a 28-day period.

• 4.The ring test showed that if the test was extended beyond 28 days no useful information could be gathered, in most cases, due to severe interferences. The blank biological oxygen demand (BOD) values were excessively high probably due to wall growth, caused by lack of agitation, and to nitrification. Thus, the recommended duration is 28 days, but if the blank BOD value remains within the 30 per cent limit (paragraphs 15 and 40) the test could be prolonged.

INFORMATION ON THE TEST SUBSTANCE

• 5.In order to know whether the test may be applied to a particular substance, some of its properties must be known. The empirical formula is required so that the theoretical oxygen demand (ThOD) may be calculated (see Appendix 3); otherwise the chemical oxygen demand (COD) of the substance must be determined to serve as the reference value. The use of COD is less satisfactory since some substances are not fully oxidised in the COD test.

• 6.The solubility of the substance should be at least 2 mg/l, though in principle less soluble substances could be tested (e.g. using ultra sonication) as could volatile substances. Information on the purity or the relative proportions of major components of the test substance is required in order that the results obtained can be interpreted, especially when the result lies close to the ‘pass’ level.

• 7.Information on the toxicity of the substance to bacteria e.g. as measured in short-term respiration tests (4) may be very useful when selecting appropriate test concentrations and may be essential for the correct interpretation of low biodegradation values. However, such information is not always sufficient for interpreting results obtained in the biodegradation test and the procedure described in paragraph 27 is more suitable.

REFERENCE SUBSTANCES

• 8.Suitable reference substances must be used to check the microbial activity of the seawater sample. Aniline, sodium acetate or sodium benzoate (for example) may be used for this purpose. A degradation of these substances of at least 60 per cent (of their ThOD) must occur within a reasonably short time span, otherwise it is recommended that the test be repeated using another seawater sample.