4-phenylbutenone

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Please refer to section "Any other information on materials and methods incl. tables" for further information

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Chemical determinations were performed for the stock solution of 200 mg/L, the samples of all test item concentrations of nominal 3.5, 1.09, 0.34, 0.10 and 0.03 mg/L and the control.
- Sampling method: Samples from the test item concentrations and the control were collected (i) from tank at exposure initiation (day 0 fresh), (ii) from tank and aquarium at renewals (day 1, day 7 day 14, day 21 and day 28 fresh and spent), (iii) from tank and aquarium at exposure termination (day 36 spent) and analysed. Fresh stock solutions were analysed at exposure initiation and at renewals.
- Sample storage conditions before analysis: not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock concentration of 200 mg/L was prepared in a glass flask with capacity of 2 L and was used to prepare the test item concentrations. An amount of 400.0 mg of the test item was weighed and mixed with 1500 mL of ISO medium (OHAUS PA 214CM/1). Resulting mixture of loading was heterogeneous with visible non-dissolved particles on the bottom. Therefore, the mixture was sonicated in the ultrasonic bath for 40 minutes (ultrasonic bath Sonic 10). Next, the content of the flask was filled up to 2L with ISO medium. Resulting stock test item concentration of 200 mg/L was homogeneous and transparent.
At exposure initiation appropriate volumes of the stock solution were added into additional aquarium with capacity of 59 L, which was darkened by black foil, in order to prepare test item concentrations, which were transferred to the test aquaria. A detailed scheme of preparing test item concentrations is presented in section "Any other information on materials and methods incl. tables".
The stock concentration of 200 mg/L was renewed daily to maintain the test item concentration as constant as possible.
- Controls: A control containing ISO medium only was tested in parallel.
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, the stock solution and test media were observed to be homogeneous and transparent.
- Other relevant information: Tanks with test item concentrations or the control, the aquarium with stock solution and the tubing of the flow-through system were darkened in order to avoid the loss of the test item due to photolytic degradation. Test solutions and the control (ISO medium) in the tanks were changed daily.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio (Hamilton) (syn. Brachydanio rerio, Cyprinus rerio)
- Source: Broodstock fish originated from the standard laboratory culture of the Łukasiewicz Research Network - Institute of Industrial Organic Chemistry, Branch Pszczyna according to the Guideline OECD 210 recommendations. The broodstock was culture in the regular aquarium of 200 L in ISO medium with constant filtration and aeration system. ISO medium was filtered through layers of thick and thin sponge, cotton wool, and ceramic refill used as mechanic filter. The infectious disease monitoring program is implemented in the animal facility at the Łukasiewicz Research Network - Institute of Industrial Organic Chemistry, Branch
Pszczyna. It provides information on animal health and hygiene in the animal facility. Before the study, a sample of zebrafish was sent to the National Veterinary Research Institute in Puławy, Poland in order to examine whether the fish were contaminated with bacterial or viral diseases. The results of examination showed that fish were free from bacterial and viral contamination. Animals from healthy batch, free from external parasites as well as visible deformations, were used as broodstock.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): not specified
- Method of collection of fertilised eggs: 24 h before the start of the experiment adult fish (broodstock) were transferred from regular aquarium of 200 L to a smaller aquarium with double bottom and artificial plants to provide shelter. The ratio of female : male fish was 2: 1 in the smaller aquarium completely shaded from light. At the test start the aquarium was exposed to light what triggered spawning. The eggs passed the first bottom of soft mesh and therefore were protected from adult fish and water turbulence.
- Subsequent handling of eggs: During the next 3 h fertilised embryos (inspected by microscopic observation) were transferred in groups of 20 into glass aquaria of 3 L volume containing the media of the test item concentrations (replicates). The embryos of the same age were used at test initiation.

POST-HATCH FEEDING
- Start date: Day 4
- Type/source of feed and frequency of feeding: Starting from day 4, feeding was introduced into every test vessel at least six times per day. The test system was fed with protozoa zooplankton (cultured for this purpose, renewed twice a week). After day 5 the larvae in each test vessel were fed with protozoa and once a day with proteins suspension. On day 12, the diet of developing test organisms was supplemented with nutritive dry feed (Ovo-Vit and Bio-Vit flakes grinded) five times per day. From day 20 larvae diet was supplemented with Artemia salina nauplii instead of proteins, once a day.
- Amount given: Test organisms in each test vessel received the same amount of food. In order to avoid accumulation of waste, surplus food and faeces were daily removed by siphoning from the bottom of each test vessel.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

36 d

Hardness:

test start: 233 - 234 mg as CaCO3/L for the test item concentration of 3.5 mg/L and the control
test end: 246 – 270 mg as CaCO3/L for the test item concentration of 1.09 mg/L and the control

Test temperature:

25 - 27 °C in individual test aquaria throughout the test

pH:

after renewal of test solutions in tanks (fresh): 7.39 – 8.40
before renewal of test solutions in tanks (aged): 7.25– 8.01

Dissolved oxygen:

after renewal of test solutions in tanks (fresh): 84 – 101% ASV
before renewal of test solutions in tanks (aged): 80 – 99% ASV

Nominal and measured concentrations:

nominal concentrations: 3.5, 1.09, 0.34, 0.10, 0.03 mg/L and a control
time-weighted mean measured concentration: 2.54, 0.623, 0.164, 0.0358, 0.0101 mg/L and a control

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass aquaria of 4 L capacities filled with 3 L volume
- Aeration: Solutions in test aquaria were not aerated till day 20 of the test. Test item concentrations and the control in the tanks were continuously aerated during the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): The flow-through design was provided by two peristaltic pumps (BT100S-1 Basic Variable-Speed Peristaltic Pump) each with twelve section heads. The flow-through system consisted of two pumps, six tanks with test item concentrations and twenty-four test aquaria placed on cuvettes per four replicates. The nominal dosing rate of the test item concentration or control medium was 6 mL/min. The test item concentration/control from tanks (aquaria capacity of 50 L) was dosed by pumps using tubing to the single replicates. The diameter of tubing was 3.1 mm.
- Renewal rate of test solution (frequency/flow rate): The flow rate was conducted with three exchange per test aquaria per 24 hours.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium (please refer to section "Any other information on materials and methods incl. tables" for further information)
- Total organic carbon: The Total Organic Carbon (TOC) determined in ISO medium sample was 1.016 mg/L.
- Culture medium different from test medium: No, the ISO medium used as diluent was the same as in the laboratory culture of zebrafish.
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Photoperiod: day : night ratio of 16 : 8 h,
- Light intensity: mean light illumination: 748 –778 lx during exposure
- Other: Constant conditions throughout the test were provided with the air-conditioning.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Hatching, survival and stages of development were observed and compared with the control.
The observations were made once a day, i.e. approximately every 24 h at the beginning of the test. During the definitive test observations for hatching, survival and symptoms of intoxication (for fertilized eggs: loss of translucency, change of coloration, for embryos, larvae: immobility and/or absence of respiratory movement and/or loss of balance and/or absence of heartbeat and/or lack of reaction to mechanical stimulus)
were conducted at exposure initiation and every 24 h of exposure thereafter.
In addition, the growth parameter wet weight, dry weight and length were determined on four test organisms per treatment group (one organism per replicate was removed) after hatching and at test termination from the mean of individual survived fish after euthanasia. Length was measured under microscope with accuracy of ± 0.1 mm (Olympus CX40 with eyepiece micrometer disc) and wet weight was determined cumulatively for four test organisms. The dry weight was determined only for the test item concentration of 0.03 mg/L. For the other concentrations and the control dry weight could not be calculated due to too low values.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: Preliminary tests according to OECD 236 were conducted first to provide details in the study plan on the treatment range and the most suitable test design. Afterwards a chronic preliminary non-GLP test according to OECD 210 in the flow-through design was performed with the test item concentrations of nominal 8.0, 1.33, and 0.22 mg/L (each was performed in two replicates per 20 embryos) and a control (in two replicates per 20 embryos) to determine a suitable concentration range for the definitive test. The flow rate was conducted with 3 exchange chambers per 24 hours.
- Results used to determine the conditions for the definitive study: Hatching started on day 2 and finished on day 5 of exposure. In the test item concentration of nominal 8 mg/L thirty-eight embryos and two larvae were dead on day 5 of exposure already. Thus, the cumulative mortality in the test item concentration of 8 mg/L was 100% on day 5. In the test item concentration of 1.33 mg/L nine larvae survived till exposure termination and the cumulative mortality resulted in 77.5%. In the test item concentration of nominal 0.22 mg/L six larvae survived till exposure termination and the cumulative mortality resulted in 85.0%. In the control hatching started on day 3 with thirty-eight embryos hatched. Hatching was completed on day 4 with forty larvae hatched. The cumulative mortality in the control finally resulted in 55.0% till exposure termination.

POST-HATCH DETAILS
- Begin of post-hatch period: Day 6

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

36 d

Dose descriptor:

EC10

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

36 d

Dose descriptor:

EC50

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Dry weight

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Dry weight

Duration:

36 d

Dose descriptor:

EC10

Effect conc.:

0.058 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Dry weight

Duration:

36 d

Dose descriptor:

EC50

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Dry weight

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

fresh weight

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

fresh weight

Duration:

36 d

Dose descriptor:

EC10

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

fresh weight

Duration:

36 d

Dose descriptor:

EC50

Effect conc.:

> 0.623 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

fresh weight

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

0.036 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

0.164 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Duration:

36 d

Dose descriptor:

EC10

Effect conc.:

0.004 mg/L

95% CI:

> 0.001 - < 0.008

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Remarks on result:

other: extrapolated (> 25% below the lowest test concentration)

Duration:

36 d

Dose descriptor:

EC50

Effect conc.:

0.065 mg/L

95% CI:

> 0.044 - < 0.095

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Key result

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

0.036 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

36 d

Dose descriptor:

EC10

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

36 d

Dose descriptor:

EC50

Effect conc.:

0.448 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Reported statistics and error estimates:

The calculations and statistical analysis were performed with a commercial software ToxRat Professional version 3.3.0.
Median concentration causing 50% reduction in hatching on day 6 (EC50/6 days on hatchability), mortality during 30 days, reduction in fresh and dry weight as well as length were calculated by probit method.
The following statistical tests were performed with hatching and post-hatch survival data during the exposure: Qualitative Trend Analysis by Contrast (Monotonicity of Concentration/Response), to check linearity followed by Tarone Test Procedure and Step-down Rao-Scott-Cochran-Armitage Test procedure.
The following statistical tests were performed with fresh weight and dry weight data during the exposure: Shapiro -Wilk’s Test for normal distribution of the data, followed by Levene’s Test on Variance Homogeneity (with Residuals) and finally Dunnet Multiply t-test Procedure.
For the parameter length the Shapiro-Wilk’s Test confirmed normal distribution of the data, followed by use of Levene’s Test on Variance Homogeneity (with Residuals) which showed that the variances were heterogeneous and finally Multiply Sequentially-rejective Welsh-t-test After Bonferroni-Holm.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD Guideline 210 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the long-term toxicity of the registered substance towards the freshwater fish Danio rerio.
The study was conducted under GLP according to OECD Guideline 210 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the long-term toxicity of the registered substance towards the freshwater fish Danio rerio.
In a flow-through test embryos of Danio rerio (zebrafish) were exposed to the test substance at nominal concentrations of 0.03, 0.10, 0.34, 1.09, 3.5 mg/L plus control for 30 days post-hatch under defined conditions. The total duration of the test was 36 days. Observations of embryos mortality and development were made every 24 h. After day 6 of the test observations of mortality of larvae, ability to acquire food, visible abnormalities in appearance for example yolk sac deformations and behaviour were made every 24 h. In addition, the growth parameter wet weight, dry weight and length were determined on four test organisms per treatment group (one organism per replicate was removed) after hatching and at test termination.
The concentrations of the test substance were chemically determined using a validated liquid chromatographic method with Diode Array Detection (HPLC-DAD). n samples collected from the tanks at exposure initiation, the determined concentrations of the test item were in the range of 116.1 – 118.0% of the nominal concentrations. The results confirm that the test item concentrations were prepared correctly. In fresh and spent samples collected from tanks and aquaria during the course of the study and at test termination, the determined concentrations of the test item were varying and not in the nominal range constantly (80-120%). Accordingly, test item concentrations could not be maintained constant throughout the test, wherefore the time weighed average concentrations (TWA) of the test material were calculated and used for the evaluation of results.
Based on the obtained biological and analytical results, the most sensitive endpoint was determined to be the 36 d NOEC for the effect parameter hatchability with a value of 0.0101 mg test item/L. The NOEC for the effect parameter post-hatch survival was determined to be 0.0358 mg/L and the NOEC for the effect parameters fresh-weight, dry-weight and length were determined to be higher than or equal to 0.623 mg/L, respectively.
Although the EC10 and EC20 values of 0.004 and 0.010 mg/L for the effect parameter post-hatch survival are below the most sensitive NOEC of 0.0101, these are not considered reliable. A post-hatch mortality of 25% is within the natural variance according to the Guideline and was not exceeded with values of 22.5 and 25% in either the control or the lowest TWA concentration of 0.0101 mg/L. With a value of 28.8%, the mortality in the second lowest concentration of 0.0358 mg/L is also only 5% higher than the control mortality, which is not considered significant according to the statistical evaluation and certainly does not correspond to a 10 or 20% effect compared to the control. The NOEC for this parameter was correctly determined to be 0.0358 mg/L respectively. Beside both values are extrapolated (even more than 25% below the lowest tested concentration in case of the EC10) and the large span of the 95% confidence intervals indicate that the endpoints are not reliable.

Executive summary:

The present study was performed under GLP according to OECD TG 210. In the definitive test Danio rerio embryos were exposed to the registered substane in a flow-through design. Embryos in the stage before cleavage the blastodisc commences were exposed to five test item concentrations of nominal 0.03, 0.10, 0.34, 1.09, 3.5 mg/L plus control for 30 days post-hatch. The total duration of the test was 36 days. The test vessels were glass aquaria with a capacity of 4 L. There were four replicates for each test item concentration and the control. Twenty embryos were introduced into each aquarium. Observations of embryos mortality and development were made every 24 h. The embryos in crystallizers were transferred from each test vessel under the stereoscopic microscope (MST 132) and observed for coagulation, loss of translucency, change of coloration, lack of heartbeat, hatching. After day 6 of the test observations of mortality of larvae, ability to acquire food, visible abnormalities in appearance for example yolk sac deformations and behaviour were made every 24 h. In addition, the growth parameter wet weight, dry weight and length were determined on four test organisms per treatment group (one organism per replicate was removed) after hatching and at test termination.

The concentrations of the test substance were chemically determined using a validated liquid chromatographic method with Diode Array Detection (HPLC-DAD).
The chemical determinations were performed for the stock solution, the samples of all test item concentrations and the control at exposure initiation, on the first day of exposure, and once a week till the exposure termination. The samples were collected from tanks and test aquaria before exchange of the test media of the test item concentrations and control (spent) and after renewal (fresh).

In samples collected from the tanks at exposure initiation, the determined concentrations of the test item were in the range of 116.1 – 118.0% of the nominal concentrations. The results confirm that the test item concentrations were prepared correctly. In fresh and spent samples collected from tanks and aquaria during the course of the study and at test termination, the determined concentrations  of the test item were varying and not in the nominal range constantly (80-120%). Accordinly, test item concentrations could not be maintained constant throughout the test, wherefore the time weighed average concentrations of the test material were calculated and use for the evaluation of results.

On the basis of the time-weighted mean of measured concentrations the following endpoint values were estimated:

Hatchability:
The EC50 value for hatchability after 6 days of exposure is 0.448 mg/L.
The EC20 value for hatchability after 6 days of exposure is 0.038 mg/L.
The EC10 value for hatchability after 6 days of exposure is 0.011 mg/L.
The LOEC value for hatchability is 0.0358 mg/L.
The NOEC value for hatchability is 0.0101 mg/L.

Post-hatch survival:
The EC50 value for survival 30 days after hatching is 0.065 mg/L (95% confidence limits: 0.044-0.095).
The EC20 value for survival 30 days after hatching is 0.010 mg/L (95% confidence limits: 0.005-0.017, extrapolated).
The EC10 value for survival 30 days after hatching is 0.004 mg/L (95% confidence limits: 0.001-0.008, extrapolated).
The LOEC value for post-hatch survival is 0.164 mg/L.
The NOEC value for post-hatch survival is 0.0358 mg/L.

Fresh weight:
The EC50, EC20 , and EC10 values could not be calculated based on obtained results due to the lack of effects.
The LOEC value for fresh weight is higher than 0.623 mg/L.
The NOEC value for fresh weight is higher than or equal to 0.623 mg/L.

Dry weight:
The EC50 value could not be calculated based on obtained results due to the lack of effects.
The EC20 value 30 days after hatching is 1.498 mg/L.
The EC10 value 30 days after hatching is 0.058 mg/L.
The LOEC value for dry weight is higher than 0.623 mg/L.
The NOEC value for dry weight is higher than or equal to 0.623 mg/L.

Length:
The EC50, EC20 , and EC10 values could not be calculated based on obtained results due to the lack of effects.
The LOEC value for length is higher than 0.623 mg/L.
The NOEC value for length is higher than or equal to 0.623 mg/L.

Description of key information

OECD 210 (GLP), Danio rerio, 36 d, flow-through, NOEC = 0.0101 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.01 mg/L

Additional information

The present study was performed under GLP according to OECD TG 210. In the definitive test Danio rerio embryos were exposed to the registered substane in a flow-through design. Embryos in the stage before cleavage the blastodisc commences were exposed to five test item concentrations of nominal 0.03, 0.10, 0.34, 1.09, 3.5 mg/L plus control for 30 days post-hatch. The total duration of the test was 36 days. The test vessels were glass aquaria with a capacity of 4 L. There were four replicates for each test item concentration and the control. Twenty embryos were introduced into each aquarium. Observations of embryos mortality and development were made every 24 h. The embryos in crystallizers were transferred from each test vessel under the stereoscopic microscope (MST 132) and observed for coagulation, loss of translucency, change of coloration, lack of heartbeat, hatching. After day 6 of the test observations of mortality of larvae, ability to acquire food, visible abnormalities in appearance for example yolk sac deformations and behaviour were made every 24 h. In addition, the growth parameter wet weight, dry weight and length were determined on four test organisms per treatment group (one organism per replicate was removed) after hatching and at test termination.

The concentrations of the test substance were chemically determined using a validated liquid chromatographic method with Diode Array Detection (HPLC-DAD).
The chemical determinations were performed for the stock solution, the samples of all test item concentrations and the control at exposure initiation, on the first day of exposure, and once a week till the exposure termination. The samples were collected from tanks and test aquaria before exchange of the test media of the test item concentrations and control (spent) and after renewal (fresh).

In samples collected from the tanks at exposure initiation, the determined concentrations of the test item were in the range of 116.1 – 118.0% of the nominal concentrations. The results confirm that the test item concentrations were prepared correctly. In fresh and spent samples collected from tanks and aquaria during the course of the study and at test termination, the determined concentrations  of the test item were varying and not in the nominal range constantly (80-120%). Accordinly, test item concentrations could not be maintained constant throughout the test, wherefore the time weighed average concentrations of the test material were calculated and use for the evaluation of results.

On the basis of the time-weighted mean of measured concentrations the following endpoint values were estimated:

Hatchability:
The EC50 value for hatchability after 6 days of exposure is 0.448 mg/L.
The EC20 value for hatchability after 6 days of exposure is 0.038 mg/L.
The EC10 value for hatchability after 6 days of exposure is 0.011 mg/L.
The LOEC value for hatchability is 0.0358 mg/L.
The NOEC value for hatchability is 0.0101 mg/L.

Post-hatch survival:
The EC50 value for survival 30 days after hatching is 0.065 mg/L (95% confidence limits: 0.044-0.095).
The EC20 value for survival 30 days after hatching is 0.010 mg/L (95% confidence limits: 0.005-0.017, extrapolated).
The EC10 value for survival 30 days after hatching is 0.004 mg/L (95% confidence limits: 0.001-0.008, extrapolated).
The LOEC value for post-hatch survival is 0.164 mg/L.
The NOEC value for post-hatch survival is 0.0358 mg/L.

Fresh weight:
The EC50, EC20 , and EC10 values could not be calculated based on obtained results due to the lack of effects.
The LOEC value for fresh weight is higher than 0.623 mg/L.
The NOEC value for fresh weight is higher than or equal to 0.623 mg/L.

Dry weight:
The EC50 value could not be calculated based on obtained results due to the lack of effects.
The EC20 value 30 days after hatching is 1.498 mg/L.
The EC10 value 30 days after hatching is 0.058 mg/L.
The LOEC value for dry weight is higher than 0.623 mg/L.
The NOEC value for dry weight is higher than or equal to 0.623 mg/L.

Length:
The EC50, EC20 , and EC10 values could not be calculated based on obtained results due to the lack of effects.
The LOEC value for length is higher than 0.623 mg/L.
The NOEC value for length is higher than or equal to 0.623 mg/L.

 

Based on the obtained biological and analytical results, the most sensitive endpoint was determined to be the 36 d NOEC for the effect parameter hatchability with a value of 0.0101 mg test item/L. The NOEC for the effect parameter post-hatch survival was determined to be 0.0358 mg/L and the NOEC for the effect parameters fresh-weight, dry-weight and length were determined to be higher than or equal to 0.623 mg/L, respectively.
Although the EC10 and EC20 values of 0.004 and 0.010 mg/L for the effect parameter post-hatch survival are below the most sensitive NOEC of 0.0101 mg/L, these are not considered reliable. A post-hatch mortality of 25% is within the natural variance according to the Guideline and was not exceeded with values of 22.5 and 25% in either the control or the lowest TWA concentration of 0.0101 mg/L. With a value of 28.8%, the mortality in the second lowest concentration of 0.0358 mg/L is also only 5% higher than the control mortality, which is not considered significant according to the statistical evaluation and certainly does not correspond to a 10 or 20% effect compared to the control. The NOEC for this parameter was correctly determined to be 0.0358 mg/L respectively. Beside both values are extrapolated (even more than 25% below the lowest tested concentration in case of the EC10) and the large span of the 95% confidence intervals indicate that the endpoints are not reliable.