Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-09-2009 to 14-09-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2007 ; signature: March 2009

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L nominal concentration
Treatment level codes: nominal: C0, C1, C2, C3, C4, C5, C6, C7 respectively.
Arithmetic Mean Measured equivalent concentrations: < 30% LOQ (control), 0.0043, 0.0068, 0.0144, 0.0298, 0.0632, 0.137 and 0.308 mg/L
LOQ = 0.00308 mg/L or 3.08 μg/L
- Sampling method: Samples of 20 mL were taken from the designated test solutions and stock solution, and transferred to a glass tube. In order to minimise losses of the test item, the water samples were extracted on site, immediately following sampling. Samples (duplicates) were taken from stock solutions (from the storage tank) and test
solutions (from the test vessels during the test) on day (immediately after preparation), end of week 1, week 2, week 3, week 4 and at end of the test from the test vessels. Selected samples were analysed of the higher concentration levels, since at these levels the biological effects were far above the NOEC; more samples were selected on a
weekly basis for lower concentrations, where it was important to relate exposure concentrations to the observed biological effects.
- Sample storage conditions before analysis: All samples (and reserve samples) designated for analysis were transferred to the test site for chemical analysis (detailed in the study report) on dry ice and were received at the analytical test site in good condition and placed in a freezer until analysis. The control reconstituted water sample was received in good condition and placed in a fridge until analysis. On the day of analysis, the samples were defrosted followed by shaking. The concentration of test item was determined by GC-MSD. The control reconstituted water sample was allowed to return to room temperature prior to extraction. The extraction procedure is described below.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in dilution water to prepared stock; serial dilution of stock to prepare test media with use of flow through system. The storage tank was filled with the desired volume of dilution water (reconstituted water) using a 10 L graduated flask. The amount of test item necessary for the desired final volume of this stock solution (“S1”, 500 μg/L nominal) was added to the dilution water, ultrasonicated for 12 to 20 minutes and thereafter stirred for at least 16 hours1) at room temperature. The storage tank was tightly closed during sonication and stirring. The stock solution in water was freshly prepared at least three times per week and used directly for preparation of test solutions. During dosing into the flow-through system (between renewals), the stock solution was stored at room temperature in the dark. During storage, the tank was closed with a lid to reduce air-exchange and evaporation. It was noted: On one occasion (day 30 to 31 of exposure) the magnetic stirrer had slipped out of the centre of the tank bottom area, therefore an exact time period of stirring could not be assigned. This batch of stock solution was ultrasonicated again for 5 minutes and stirred for 2 additional hours before use. The flow-through system was adjusted to produce the test concentrations. The actually delivered concentrations were calculated based on the mean measured volumes of reconstituted water and stock solution delivered by the flow-through system per run. One day before start of exposure, the flow-through system was started in order to condition the flow-through equipment. The number of runs per day was adjusted to produce the nominal flow rates. The nominal flow rate was 1.5 L/vessel/day (5 vessel volume exchanges/day). On day 30: the nominal flow rate was increased to 3.0 L/vessel/day (10 vessel volume exchanges/day). The mean total recovery for all measured test solutions was 50.6% (range 43.8 to 61.5%) of the nominal concentration. Procedural recoveries were 81 to 108%. Therefore the biological results are based on nominal as well as on arithmetic mean measured concentrations for each individual test concentration throughout the exposure period.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra Fish (Danio rerio)
- Strain: Hamilton-Buchanan 1822
- Source: in house laboratory cultures (historic information available in the full study report).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): 2 replicates of 30 fertilized eggs per concentration level.
- Method of collection of fertilised eggs: On the evening before test start, glass bowls covered with stainless steel mesh were introduced in the holding tanks of the adult zebra fish. Water plants (Microsorum pteropus) were placed on the mesh, allowing the fish to spawn. The spawning bowls were removed from the holding tanks on the day of test start shortly after onset of illumination, which triggers fertilization. The content of the bowls was poured over a sieve (mesh size 0.5 mm), rinsed with reconstituted water and collected in a glass vessel filled with reconstituted water.
- Subsequent handling of eggs: groups of 10 - 20 eggs were transferred to glass dishes containing test solution of each designated exposure vessel (acclimated at test temperature), until all dishes contained approximately 130 eggs. The dishes were placed into an incubator set to 25°C for two hours. Unfertilized and damaged eggs were removed and the number of eggs per dish was reduced to 30. Subsequently the eggs were transferred to the exposure vessels.

POST-HATCH FEEDING
- Start date: On the day after the first larva per test vessel was recorded to swim up (day 4 of exposure), feeding was started by adding food to the vessels
- Type/source of feed: . The test organisms were fed dry food (NovoBaby 01 and 02, Tetra), live Artemia nauplius larvae and parametia (Paramecium caudatum)
- Amount given: ad libitum; the daily ration was fed in 3 - 4 equal portions on workdays. On weekends the daily ration was fed in 2 - 3 portions. Uneaten food was removed at least once daily. The food ration was adjusted to the number of living fish per test vessel. Food was withheld from the fish for 24 hours prior to test end.
- Frequency of feeding: See above.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Remarks on exposure duration:

In accordance with the OECD TG 210 guideline.

Post exposure observation period:

The test period (exposure of test organisms to the test solutions) was 36 days (guideline specifies > 30 days).

Hardness:

Min: 157, Max: 182 mg/L as CaCO3

Test temperature:

Min: 24.1°C, Max: 25.8°C
On-line temperature measurement in one test vessel recorded every 30 minutes from day 0 to day 36: Min: 24.8°C, Max: 25.5°C

pH:

Min: 7.0, Max: 7.5

Dissolved oxygen:

Dissolved oxygen concentration: Min: 6.0 mg/L, Max: 9.1 mg/L; equivalent to Min: 74%, Max: 110% of air saturation value (ASV)
It was noted: on one occasion, (day 30 of exposure), oxygen concentrations below 60% of the air saturation value (ASV) were measured in the vessels at 0, 8, 16, and 31 μg/L. After doubling the flow rate, the oxygen concentration was measured again in the respective treatments, and was above 80% of the ASV. Since observations of the test organisms did not indicate any impairment in these vessels, and since the oxygen content recorded on all other occasions was above 70% of ASV, it was considered this had no impact on the integrity of the study.

Conductivity:

763 to 790 μS/cm (testing of reconstituted water)

Nominal and measured concentrations:

0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L nominal concentration
Arithmetic Mean Measured equivalent concentrations: < 30% LOQ (control), 0.0043, 0.0068, 0.0144, 0.0298, 0.0632, 0.137 and 0.308 mg/L
LOQ = 0.00308 mg/L or 3.08 μg/L

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): On the evening before test start, glass bowls covered with stainless steel mesh were introduced in the holding tanks of the adult zebra fish. Water plants (Microsorum pteropus) were placed on the mesh, allowing the fish to spawn. The spawning bowls were removed from the holding tanks on the day of test start shortly after onset of illumination, which triggers fertilization. The content of the bowls was poured over a sieve (mesh size 0.5 mm), rinsed with reconstituted water and collected in a glass vessel filled with reconstituted water. Groups of 10 - 20 eggs were transferred to glass dishes containing test solution of each designated exposure vessel (acclimated at test temperature), until all dishes contained approximately 130 eggs. The dishes were placed into an incubator set to 25°C for two hours. Unfertilized and damaged eggs were removed and the number of eggs per dish was reduced to 30. Subsequently the eggs were transferred to the exposure vessels.
- Test vessel: Glass vessels, 0.5 L volume
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Diameter: 14 cm, Height: 6 cm; vessels were fitted with a meshed overflow: center of outflow: 3.5 cm, diameter of outflow: 1 cm. The vessels were covered with a transparent lid to reduce air-exchange and evaporation. Fill volume: ca. 0.3 L.
- Aeration: No. Flow through experiment.
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic
- Renewal rate of test solution (frequency/flow rate): The flow-through system was adjusted to produce the test concentrations. The actually delivered concentrations were calculated based on the mean measured volumes of reconstituted water and stock solution delivered by the flow-through system per run. One day before start of exposure, the flow-through system was started in order to condition the flow-through equipment. The number of runs per day was adjusted to produce the nominal flow rates. The nominal flow rate was 1.5 L/vessel/day (5 vessel volume exchanges/day). On day 30: the nominal flow rate was increased to 3.0 L/vessel/day (10 vessel volume exchanges/day).
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: 30 organisms per vessel.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water according to OECD guideline No. 203 was used to dilute the test item and to keep the fish before and during the period of the test.
- Total organic carbon: Local tapwater is treated by reverse osmosis and ion exchange to prepare deionised water. Therefore a contamination with heavy metals, pesticides and TOC can be excluded.
- Particulate matter: See above.
- Metals: See above.
- Pesticides: See above.
- Chlorine: See above.
- Alkalinity: Not reported.
- Ca/mg ratio: CaCl2.2 H2O 294.0 mg/L / MgSO4.7H2O 123.0 mg/L
- Salinity: Reconstituted water was diluted with deionised water (1:1; v/v), and supplemented with 1% of artificial seawater
- Culture medium different from test medium: No.
- Intervals of water quality measurement: See above.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 12/12 hours light/dark
- Light intensity: 180 – 331 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : hatching success ; post-hatch success and survival ; weight and length of survivors ; behaviour and morphology

VEHICLE CONTROL PERFORMED: No. Not applicable.

RANGE-FINDING STUDY
- Test concentrations: No. Definitive test: based on preceding acute toxicity study (information in the full study report) and/or decision of the study monitor and study director.
- Results used to determine the conditions for the definitive study: Not applicable.

POST-HATCH DETAILS
- Begin of post-hatch period: day 3, to day 10 (conclusion of hatching)
- No. of hatched eggs (alevins)/treatment released to the test chamber: 2 replicates of 30 (60 total)
- Release of alevins from incubation cups to test chamber on day no.: Not applicable.

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 60 (in 2 replicates of 30)
- Removal of eggs to check the embryonic development on day no.: Not applicable. Fish were examined for survival, behaviour, morphology, length, weight on day 36.

Reference substance (positive control):

no

Duration:

30 d

Dose descriptor:

NOEC

Remarks:

hatching success

Effect conc.:

0.137 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks:

hatching success

Duration:

30 d

Dose descriptor:

LOEC

Remarks:

hatching success

Effect conc.:

0.308 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks:

hatching success

Duration:

30 d

Dose descriptor:

NOEC

Remarks:

mortality (post-hatch success)

Effect conc.:

0.004 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch success

Duration:

30 d

Dose descriptor:

LOEC

Remarks:

mortality (post-hatch success)

Effect conc.:

0.007 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch success

Duration:

30 d

Dose descriptor:

EC50

Remarks:

mortality (post-hatch success)

Effect conc.:

0.007 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch success

Duration:

30 d

Dose descriptor:

NOEC

Remarks:

healthy fish

Effect conc.:

>= 0.137 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks:

(healthy fish)

Duration:

30 d

Dose descriptor:

NOEC

Remarks:

dry body weight

Effect conc.:

>= 0.137 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: See table.
- Days to hatch or time to release of young: The fish started hatching on day 3 after start of exposure at C2 to C7, while in the control and at C1, hatch started on day 4. Hatch was finished on day 9 to 10 for all treatments. A concentration-response relationship could not be observed.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: In controls the hatching success was 100%. In all treatments up to 250 μg/L the hatching success range between 86.7% and 98.4%. At a concentration of 500 μg/L the hatching success was 58.4%.
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): See table.
- Observations on body length and weight of young and/or exposed parents at one or more time periods: Statistically significant differences of weight were not observed between treated and control groups at p ≤ 0.05. No correlation was observed between the concentration of the test item and the dry weight of fish. Statistically significant differences of length were not observed between treated and control groups at p ≤ 0.05. No correlation was observed between the concentration of the test item and the length of fish.
- Number of healthy fish at end of test: In the controls, the mean post-hatch success was 70.0%. In the treatments, the per treatment post-hatch success was 61.0%, 39.7%, 38.2%, 33.42.7%, 9.1%, 5.3% and 0% at 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 μg/L (nominal), respectively.
- Type of and number with morphological abnormalities: None.
- Type of and number with behavioural abnormalities: None.
- Type and number of developmental effects: None.
- Type and magnitude of hormonal changes: Not applicable.
- Other biological observations: None.
- Effect concentrations exceeding solubility of substance in test medium: The mean total recovery for all measured test solutions was 50.6% (range 43.8 to 61.5%) of the nominal concentration. Procedural recoveries were 81 to 108%. Therefore the biological results are based on nominal as well as on arithmetic mean measured concentrations for each individual test concentration throughout the exposure period. . It can be speculated the differences in nominal/measured test concentrations was due to possible: adsorption and biological effects and/or to any food/organic waste matter present in the test system.
- Incidents in the course of the test which might have influenced the results: No.

Reported statistics and error estimates:

The following biological parameters were evaluated statistically in comparison to the control fish where the data allowed such comparisons:
hatching success, - mortality (post-hatch success) and numbers of healthy fish by: probit analysis (Fisher's Exact Binomial Test with Bonferroni Correction)
dry weight and/or length of the surviving fish, per treatment means by: Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment
ToxRat 2.10 Professional was used for statistical analysis

Table 1.0 : Summary of biological results

Treatment level codes	Nominal Concentration /mg/L	Arithmetic mean measured concentration /mg/L	Introduced eggs	Dead before hatch	hatched	Dead fish after hatch	Total mortality	Survivors	Deformed fish / abnormal behaviour	Healthy fish
C0	0 (control)	< 30% LOQ	60	0	60	18	18	42	0	42
C1	0.0078	0.0043	60	1	59	23	24	36	0	36
C2	0.0156	0.0068	60	2	58	35	37	23	0	23
C3	0.0313	0.0144	60	5	55	34	39	21	0	21
C4	0.0625	0.0298	60	8	52	35	43	17	0	17
C5	0.125	0.0632	60	5	55	50	55	5	0	5
C6	0.25	0.137	60	3	57	54	57	3	0	3
C7	0.50	0.308	60	25	35	35	60	0	0	0

LOQ = 0.00308 mg/L or 3.08 μg/L

Validity criteria fulfilled:

yes

Conclusions:

The test item 30d-NOEC (mortality) based on post hatch success and/or healthy fish behaviour/morphology was 0.0043 mg/L and the 30d-LOEC was 0.0068 mg/L. All effects were based on arithmetic mean measured concentrations.

Executive summary:

The early-life-stage toxicity to Zebrafish (Danio rerio), was carried out according to OECD TG 210 guideline under GLP. A definitive test was conducted on 60 eggs per concentration. On the evening before test start, glass bowls covered with stainless steel mesh were introduced in the holding tanks of the adult zebra fish. Water plants (Microsorum pteropus) were placed on the mesh, allowing the fish to spawn. The spawning bowls were removed from the holding tanks on the day of test start shortly after onset of illumination, which triggers fertilization. The content of the bowls was poured over a sieve (mesh size 0.5 mm), rinsed with reconstituted water and collected in a glass vessel filled with reconstituted water. Groups of 10 - 20 eggs were transferred to glass dishes containing test solution of each designated exposure vessel (acclimated at test temperature), until all dishes contained approximately 130 eggs. The dishes were placed into an incubator set to 25°C for two hours. Unfertilized and damaged eggs were removed and the number of eggs per dish was reduced to 30 (per replicate). Subsequently the eggs were transferred to the exposure vessels. The exposures were conducted at nominal concentrations of: 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L. Exposure conditions were prepared by dilution from stock solution. The storage tank was filled with the desired volume of dilution water (reconstituted water) using a 10 L graduated flask. The amount of test item necessary for the desired final volume of this stock solution (“S1”, 500 μg/L nominal) was added to the dilution water, ultrasonicated for 12 to 20 minutes and thereafter stirred for at least 16 hours) at room temperature. The storage tank was tightly closed during sonication and stirring. The stock solution in water was freshly prepared at least three times per week and used directly for preparation of test solutions. During dosing into the flow-through system (between renewals), the stock solution was stored at room temperature in the dark. During storage, the tank was closed with a lid to reduce air-exchange and evaporation. Total test duration was 36 days. It was noted: On one occasion (day 30 to 31 of exposure) the magnetic stirrer had slipped out of the centre of the tank bottom area, therefore an exact time period of stirring could not be assigned. This batch of stock solution was ultrasonicated again for 5 minutes and stirred for 2 additional hours before use. The flow-through system was adjusted to produce the test concentrations. The actually delivered concentrations were calculated based on the mean measured volumes of reconstituted water and stock solution delivered by the flow-through system per run. One day before start of exposure, the flow-through system was started in order to condition the flow-through equipment. The number of runs per day was adjusted to produce the nominal flow rates. The nominal flow rate was 1.5 L/vessel/day (5 vessel volume exchanges/day). On day 30: the nominal flow rate was increased to 3.0 L/vessel/day (10 vessel volume exchanges/day). The chemical analysis indicated the mean total recovery for all measured test solutions was 50.6% (range 43.8 to 61.5%) of the nominal concentration. Procedural recoveries were 81 to 108%. Therefore the biological results are based on nominal as well as on arithmetic mean measured concentrations for each individual test concentration throughout the exposure period. The nominal concentrations were : 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L and the equivalent arithmetic mean measured concentrations were : < 30% LOQ (control), 0.0043, 0.0068, 0.0144, 0.0298, 0.0632, 0.137 and 0.308 mg/L. The LOQ was 0.00308 mg/L or 3.08 μg/L. All validity criteria were considered to have been met or with acceptable minor deviations that did not impact the integrity of the study. The test item 30d-NOEC (hatching success and length/bodyweight) was 0.137 mg/L. The 30d-NOEC based on mortality: from post hatch success and/or healthy fish behaviour/morphology was 0.0043 mg/L and the 30d-LOEC was 0.0068 mg/L. All effects were based on arithmetic mean measured concentrations.

Description of key information

NOEC (fish : mortality) : 0.0043 mg/L based on arithmetic mean measured concentrations, 36-days freshwater, OECD TG 210, 2010

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.004 mg/L

Additional information

Key study : OECD TG 2010, 2010 : The early-life-stage toxicity to Zebrafish (Danio rerio), was carried out according to OECD TG 210 guideline under GLP. A definitive test was conducted on 60 eggs per concentration. On the evening before test start, glass bowls covered with stainless steel mesh were introduced in the holding tanks of the adult zebra fish. Water plants (Microsorum pteropus) were placed on the mesh, allowing the fish to spawn. The spawning bowls were removed from the holding tanks on the day of test start shortly after onset of illumination, which triggers fertilization. The content of the bowls was poured over a sieve (mesh size 0.5 mm), rinsed with reconstituted water and collected in a glass vessel filled with reconstituted water. Groups of 10 - 20 eggs were transferred to glass dishes containing test solution of each designated exposure vessel (acclimated at test temperature), until all dishes contained approximately 130 eggs. The dishes were placed into an incubator set to 25°C for two hours. Unfertilized and damaged eggs were removed and the number of eggs per dish was reduced to 30 (per replicate). Subsequently the eggs were transferred to the exposure vessels. The exposures were conducted at nominal concentrations of: 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L. Exposure conditions were prepared by dilution from stock solution. The storage tank was filled with the desired volume of dilution water (reconstituted water) using a 10 L graduated flask. The amount of test item necessary for the desired final volume of this stock solution (“S1”, 500 μg/L nominal) was added to the dilution water, ultrasonicated for 12 to 20 minutes and thereafter stirred for at least 16 hours) at room temperature. The storage tank was tightly closed during sonication and stirring. The stock solution in water was freshly prepared at least three times per week and used directly for preparation of test solutions. During dosing into the flow-through system (between renewals), the stock solution was stored at room temperature in the dark. During storage, the tank was closed with a lid to reduce air-exchange and evaporation. Total test duration was 36 days. It was noted: On one occasion (day 30 to 31 of exposure) the magnetic stirrer had slipped out of the centre of the tank bottom area, therefore an exact time period of stirring could not be assigned. This batch of stock solution was ultrasonicated again for 5 minutes and stirred for 2 additional hours before use. The flow-through system was adjusted to produce the test concentrations. The actually delivered concentrations were calculated based on the mean measured volumes of reconstituted water and stock solution delivered by the flow-through system per run. One day before start of exposure, the flow-through system was started in order to condition the flow-through equipment. The number of runs per day was adjusted to produce the nominal flow rates. The nominal flow rate was 1.5 L/vessel/day (5 vessel volume exchanges/day). On day 30: the nominal flow rate was increased to 3.0 L/vessel/day (10 vessel volume exchanges/day). The chemical analysis indicated the mean total recovery for all measured test solutions was 50.6% (range 43.8 to 61.5%) of the nominal concentration. Procedural recoveries were 81 to 108%. Therefore the biological results are based on nominal as well as on arithmetic mean measured concentrations for each individual test concentration throughout the exposure period. The nominal concentrations were : 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L and the equivalent arithmetic mean measured concentrations were : < 30% LOQ (control), 0.0043, 0.0068, 0.0144, 0.0298, 0.0632, 0.137 and 0.308 mg/L. The LOQ was 0.00308 mg/L or 3.08 μg/L. All validity criteria were considered to have been met or with acceptable minor deviations that did not impact the integrity of the study. The test item 30d-NOEC (hatching success and length/bodyweight) was 0.137 mg/L. The 30d-NOEC based on mortality: from post hatch success and/or healthy fish behaviour/morphology was 0.0043 mg/L and the 30d-LOEC was 0.0068 mg/L. All effects were based on arithmetic mean measured concentrations.