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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Febraury 2019 to 20 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines (OECD 210) and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Analytical data are required by the guidelines for verification of test item concentrations as well as the stability of the test item over the entire test period. Analytical samples (5 mL) were taken from all replicates at all test item concentrations and control at test start. Weekly samples from every replicate of each treatment were taken. Additionally, samples (500 µL) were taken from each individual stock solution at day of preparation (0d fresh, 7d fresh and aged, 14d fresh and aged, 21d fresh and aged, 28d fresh and aged) and at day of the next preparation (aged; usually after 7 days) and at test end (aged).
All samples were stored deep frozen until they were transferred to the analytical laboratory.
Vehicle:
yes
Remarks:
Dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Eluate:
- Differential loading:
- Controls: Negative and solvent
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Stock solution = 100 % DMF, final test solutions 0.06 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
- Other relevant information: The quantities of test item required for preparing all stock solutions were weighed on a weighing scoop and transferred to a glass bottle. The stock solutions were prepared one day prior to test start (introduction of fertilised eggs) by dilution of the test item in 100 mL dimethylformamide and thereafter once a week, each Wednesday. The stability of the test item in the stock solutions during the renewal period was confirmed in a non-GLP pre-experiment. The stock solutions were stored in 100 mL glass bottles, shielded against light. For each replicate of each concentration, 0.120 mL of the appropriate stock solution was taken from these bottles with a syringe pump and transferred to a mixing tank, where it was mixed with 2000 mL test medium applied by a time-controlled valve. From here on, the fresh test solutions were transferred from the mixing tank to the testing chambers by means of PTFE tubes.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra Danio
- Strain: Wildstrain
- Source: Fraunhofer IME (Schmallenberg, Germany)
- Feeding during test: Suspension of artificial fry food consisting of commercial fry dust food (Caviar 50-100 µm, AquaBern, Belgium) and rotifers (AquaHobby Fischfutterhandel, Germay; purchased frozen; after unfreezing washed and grinded); later a suspension of commercial fry food (Caviar 100-200 µm, AquaBern, Belgium) and grinded commercial fish food (TetraMin, Tetra GmbH, Germany); fed 3 - 7 times a day, from day 6 onwards. At day 5, fish were fed three times a day. Additionally, newly hatched brine shrimps were fed ad libitum. Test vessels were daily checked on surplus food which was removed, if necessary.

ACCLIMATION
The fish were cultured in a climate-controlled room with 16 hours of illumination and 8 hours of darkness, in groups of approximately of 100 – 400 fish in ISO test water. In the two weeks prior to the test start:
• The pH-value of the culturing water was within a range of 6.0 – 8.5.
• The dissolved oxygen was above 60 % of the air saturation.
• The fish were cultured at a temperature of 25 ± 2 °C in a climate-controlled room with 16 hours of illumination and 8 hours of darkness.
• The animals were fed with commercial fish flake food (TetraMin, Tetra GmbH, Germany) and protein-enriched pellet food (Caviar, AquaBern, Belgium) at least once per day.
Eggs were produced by group spawning. To stimulate egg production spawning trays were placed in the culturing tanks directly after turning on the light in the morning. After 30 min of spawning, the spawning trays were removed and eggs were collected.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Remarks on exposure duration:
Consistent with OECD 210 Guideline requirements
Hardness:
250 mg/L CaCO3
pH:
The mean pH-value of the control was determined to be 7.51 +/- 0.15, the mean temperature of all treatments was measured to be 25.6 +/- 0.3 °C
Dissolved oxygen:
Dissolved oxygen concentration was determined to be 89 +/- 11 % of the air saturation.
Conductivity:
696 µS/cm
Nominal and measured concentrations:
Nominal Concentrations: 0.0060, 0.019, 0.060, 0.19 and 0.60 mg/L (including solvent & negative control)
Measured Concentrations: 0.00384, 0.0122, 0.0330, 0.108 and 0.330 mg/L (arithmetic mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass chamber
- Size of vessel: 14 L
- Material, size, headspace, fill volume: Glass, including breeding chambers which were glass cylinders closed with stainless steel mesh at the bottom, fill volume per glass chamber = 8 L
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic pump
- Renewal rate of test solution (frequency/flow rate): 6 test chamber volumes per 24 hours
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: N/A

TEST MEDIUM / WATER PARAMETERS
reconstituted test water (ISO water; OECD test guideline 203 (1992)) consisting of analytical grade salts dissolved in purified water:
CaCl2.2H2O = 294 mg/L
MgSO4.7H2O = 123 mg/L
NaHCO3 = 64.8 mg/L
KCl = 5.75 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light : 8h dark - with 15 min transition periods
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Hatching and mortality of the fish were observed daily and the numbers recorded. Dead embryos, larvae or juveniles were removed as soon as observed and recorded. Hatching success was calculated by dividing the number of successfully hatched larvae in a replicate at day 6 by the number of eggs added to that replicate. For post-hatch mortality the number of surviving fish at test end was divided by the number of successfully hatched larvae at day 6. Cumulative mortality (including the number of dead eggs) was calculated by subtracting the number of surviving fish at test end from the number of eggs at the start of the test (80/treatment).
Daily records were made of visibly abnormal appearance and behaviour of the test fish. These included hyperventilation, loss of equilibrium, atypical quiescence, swimming behaviour, atypical feeding behaviour and phenotypic malformations.
The weight and total length of each fish were determined at the end of the test. Total length was obtained from a photograph taken after the fish had been euthanized, using the image analysis software ImageJ 1.50i (Rasband). Fresh weight was measured for each individual fish after blotting dry. Dry weight was determined, after 192 h of drying at 60 °C, by pooling all fish of each treatment replicate and dividing the overall dry weight per replicate by the number of fish in that replicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 0.0100, 0.100 and 1.00 mg/L and a solvent control
- Results used to determine the conditions for the definitive study: Yes
Duration:
35 d
Dose descriptor:
EC10
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
not determinable
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.33 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
0.085 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.108 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.108 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
0.281 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.033 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
0.019 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
Fresh weight
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
Fresh weight
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
0.043 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
Dry weight
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.033 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
Dry weight
Details on results:
- Hatching and Mortality:
According to statistical calculations, no effects on hatching could be detected throughout the test period. Regarding cumulative mortality and post-hatch survival, a significant increase in mortality was observed between the highest test concentration and pooled controls. The mean hatching success in the pooled controls and the treatment groups ranged from 97.5 to 100 %, while cumulative mortality ranged from 5.0 to 42.5 %. All fish hatched within day 3 to 8 after test start.

- Observations:
At the concentration levels up to and including 0.060 mg/L single fish of smaller size or with behavioural abnormalities were recorded. However, these minor alterations at generally less than 10 % of the fish are not untypical during the fish development in an early-life stage test and are therefore not assessed to be caused by a toxic effect of the test item.
At 0.190 mg/L, many fish seemed to develop more slowly than in lower test concentrations and control from 9 days onwards. After 27 days, all fish seemed to be generally smaller than in the controls.
At 0.60 mg/L, eggs generally displayed a slight hatching delay in one replicate. Many fish displayed abnormal swimming behaviour and only moved after stimulation during the first 8 days of the test. From 9 days onwards, many fish with slower development were observed (i.e. smaller than in the lower test concentrations and control). At the same time, some individuals were observed with reduced activity at the bottom of the test vessels, or only showing signs of life after stimulation. An increased mortality rate could further be observed. Many fish seemed generally smaller than in the controls towards test end.

- Body Weight and Length:
On dry weight and total length of fish at test end, no statistically significant influence could be detected at the concentrations up to and including 0.060 mg/L. At 0.190 and 0.600 mg/L a statistically significant reduction was detected. Regarding fresh weight, no statistically significant influence could be detected at concentrations 0.0060 mg/L. For all remaining test concentrations, a significant reduction was observed.
Reported statistics and error estimates:
The EC10, EC20 and EC50 are the test item concentrations causing 10, 20 and 50 % effect on the test organisms compared to the control. The LOEC is the lowest test item concentration tested showing a statistically significant effect (p < 0.05) when compared to the pooled control. However, all test concentrations above the LOEC should have a harmful effect equal to or greater than those observed at the LOEC. The NOEC is the test item concentration immediately below the LOEC, which when compared with the pooled control, has no statistically significant effect (p < 0.05).
For the calculation of ECx, NOEC and LOEC, the statistical program ToxRat Professional 3.3.0 was used. The replicates were the unit of analysis, i.e. the mean values of each replicate were used for the data evaluation of each test concentration. No significant differences were observed between the control and solvent control. Both controls were therefore used for statistical analysis.

Hatching, mortality, mean length and body weight of test organisms at termination of the test (day 35)

Amyl Cinnamic Aldehyde nominal

Hatching success

(day 6)

Post-hatch survival

Cumulative mortality

Mean fish length

Mean fish fresh weight

Mean fish

dry weight

[mg/L]

[%]

[%]

[%]

[mm]

[mg]

[mg]

Control

97.5

96.2

6.3

19.8

70.9

19.8

Solvent

98.8

94.9

6.3

20.2

73.4

20.8

0.0060

97.5

97.4

5.0

19.8

68.3

19.4

0.0190

100.0

92.5

7.5

19.7

64.8

19.4

0.0600

100.0

91.3

8.8

19.9

66.1

19.8

0.190

98.8

94.9

6.3

18.5

56.8

16.8

0.600

98.8

58.2

42.5

17.8

50.8

15.4

Measured concentrationsofAmyl Cinnamic Aldehydein the test solutionsduring the test

Amyl Cinnamic Aldehyde

nominal

[mg/L]

Amyl Cinnamic Aldehyde

nominal
[mg a.i./L]

Sampling

Amyl Cinnamic Aldehyde found

Arithmetic mean Measured

[%]

Amyl Cinnamic Aldehyde

measured

[mg/L]

[mg a.i./L]

[% of nominal]

Control

-

0 d fresh Rep. 2

0.000328

-

-

 

0 d fresh Rep. 1

0.000224

-

0 d fresh Rep. 4

0.000332

-

7 d fresh Rep. 2

0.000332

-

21 d fresh Rep. 1

0.0000489

< LOD

35 d aged Rep. 3

0.000124

< LOQ

Solvent control

-

0 d fresh Rep. 2

0.000528

-

-

 

0 d fresh Rep. 1

0.000193

< LOQ

0 d fresh Rep. 4

0.000279

-

7 d fresh Rep. 2

0.000388

-

21 d fresh Rep. 1

0.0000323

< LOQ

35 d aged Rep. 3

0.0000937

< LOQ

0.0060

0.00591

0 d fresh Rep. 2

0.00545

92

64

0.003841)

7 d fresh Rep. 3

0.00449

76

14 d fresh Rep. 4

0.00451

76

21 d fresh Rep. 1

0.00260

44

28 d fresh Rep. 2

0.00316

53

35 d aged Rep. 3

0.00239

40

0.0190

0.0187

0 d fresh Rep. 2

0.0175

94

64

0.01221)

7 d fresh Rep. 3

0.0153

82

14 d fresh Rep. 4

0.0149

80

21 d fresh Rep. 1

0.00835

45

28 d fresh Rep. 2

0.00891

48

35 d aged Rep. 3

0.00696

37

0.0600

0.0591

0 d fresh Rep. 2

0.0499

84

55

0.03301)

7 d fresh Rep. 3

0.0402

68

14 d fresh Rep. 4

0.0404

68

21 d fresh Rep. 1

0.0267

45

28 d fresh Rep. 2

0.0240

41

35 d aged Rep. 3

0.0152

26

0.190

0.187

0 d fresh Rep. 2

0.169

90

57

0.1081)

7 d fresh Rep. 3

0.130

70

14 d fresh Rep. 4

0.158

84

21 d fresh Rep. 1

0.0566

30

28 d fresh Rep. 2

0.0756

40

35 d aged Rep. 3

0.0541

29

0.600

0.591

0 d fresh Rep. 2

0.461

78

55

0.3301)

7 d fresh Rep. 3

0.362

61

14 d fresh Rep. 4

0.392

66

21 d fresh Rep. 1

0.238

40

28 d fresh Rep. 2

0.252

43

35 d aged Rep. 3

0.238

40

 

LOQ = 0.000200 mg a.i./L; LOD = 0.0000600 mg a.i./L

Arithmeticmean measured concentration

Validity criteria fulfilled:
yes
Conclusions:
According to the results of the test, the overall NOEC (35 d) was determined to be 0.00384 mg/L Amyl Cinnamic Aldehyde (measured). The overall LOEC (35 days) was determined to be 0.0122 mg/L Amyl Cinnamic Aldehyde (measured). The EC10 (35 days) values of the most sensitive parameter (fresh weight) was determined to be 0.019 mg/L Amyl Cinnamic Aldehyde (measured). The EC20 (35 days) values of the most sensitive parameter (fresh weight) was determined to be 0.105 mg/L Amyl Cinnamic Aldehyde (measured). No EC50-values could be statistically determined.
Executive summary:

OECD 210 (2019)

The objective of the study was to assess the effects of the test item on the early-life stages of Zebrafish Danio rerio and the determination of EC10, 20, 50 values, the lowest observed effect concentration (LOEC) and hence, the no observed effect concentration (NOEC), where possible.

Materials & Methods

Test item: Amyl Cinnamic Aldehyde, batch number: 51757: (2E)-3-phenyl-2-pentyl-prop-2-enal, purity (analysed): 98.43 % w/w.

Test species: Danio rerio Hamilton (Cypriniformes: Cyprinidae).

Test design: The test was performed as a concentration-response test in a flow-through design with a duration of 35 days and renewal of stock solutions once per week, every Wednesday. Test solutions were prepared by dilution of the Test item in dimethylformamide (DMF) and application of the stock solutions into mixing chambers before being transferred to the testing chambers by means of PTFE tubes once per week.  Flow-rate was equivalent to 6 test chamber volumes per 24 hours.

For the test concentrations and the control 80 fertilised eggs (divided in four groups of 20 eggs each) were used.

Hatching and mortality of the test fish were observed daily. Records were made on mortality and visible abnormal appearance and behaviour of fish. Length, wet and dry weight of the surviving fish were measured at test end. Analytical determinations of Amyl Cinnamic Aldehyde content in test solutions were regularly conducted.

Test rates: Following a flow-through non-GLP range-finding test with nominal test item concentrations of 0.0100, 0.100 and 1.00 mg/L and a solvent control, a main test with nominal concentrations of 0.0060, 0.019, 0.060, 0.19 and 0.60 mg/L, control and solvent control was performed.

Endpoints: EC10, 20, 50, LOEC (Lowest Observed Effect Concentration) and the NOEC (No Observed Effect Concentration).

Test conditions: Temperature, pH-value and dissolved oxygen saturation of the test solutions were measured every seven days in test solutions in all test replicates (including test start and test end). Temperature was also monitored continuously in one control vessel. Water hardness was measured at the beginning of the test and at test end in vessel of each treatment. Light intensity was measured over four representative aquaria.

Samples analysed: At test start, three analytical samples taken from control and solvent control were analysed. Additionally, samples from one replicate were analysed for all test item concentrations. At days 7 and 14, 21, 28 and 35, one sample was analysed for all test item concentrations. Additionally, one sample was measured for the control and solvent control after 7, 21 and 35 days.

Statistics: All statistical calculations were carried out using the software-program ToxRat 3.3.0. ECx, NOEC and LOEC were determined by using an appropriate multiple comparison method.

Results

Validity criteria: Control survival and post-hatch success: In the control the overall survival of fertilised eggs should be greater or equal to 70 % and post-hatch success should be greater or equal to 75 %. In this study, the control survival of fertilized eggs was 97.5 % and post-hatch success was 96.2 %. The survival of fertilized eggs in the solvent control was 98.8 %, with a post-hatch success of 94.9 %.

Oxygen saturation: The dissolved oxygen concentration in control and test vessels should be at least 60 % of the air saturation value throughout the test. In this test, the dissolved oxygen concentration was ≥ 62 % % of air saturation throughout the test.

Water temperature: The water temperature should not differ by more than  1.5 °C between the test vessels or between successive days at any time during the test and should be within the temperature range of 26.0  1.5 °C. In this study, the water temperature validity criteria were fulfilled.

Test concentrations: The analytical measure of the test concentrations is compulsory.

Test conditions: The total hardness (as CaCO3) of the water was determined to be 14°dH corresponding to 250 mg/L CaCO3. The mean pH-value of the control was determined to be 7.51  0.15, the mean temperature of all treatments was measured to be 25.6  0.3 °C and the oxygen concentration was determined to be 89  11 % of the air saturation.

Analytical Results: The initial measured content of test item was between 78 and 94 % of nominal and the aged measured content at test end (day 35) was between 26 and 48 % of nominal. Since the content of test item in the samples was not between 80 and 120 % of nominal throughout the entire study, the toxicological endpoints were evaluated using arithmetic mean measured concentrations.

Statistical Results: ECx, NOEC and LOEC-values of fish early-life stages exposed to the Amyl Cinnamic Aldehyde (based on measured concentrations)

Conclusions

According to the results of the test, the overall NOEC (35 d) was determined to be 0.00384 mg/L Amyl Cinnamic Aldehyde (measured). The overall LOEC (35 days) was determined to be 0.0122 mg/L Amyl Cinnamic Aldehyde (measured). The EC10 (35 days) values of the most sensitive parameter (fresh weight) was determined to be 0.019 mg/L Amyl Cinnamic Aldehyde (measured). The EC20 (35 days) values of the most sensitive parameter (fresh weight) was determined to be 0.105 mg/L Amyl Cinnamic Aldehyde (measured). No EC50-values could be statistically determined.

Description of key information

35d EC10 (fresh weight) = 0.019 mg/L; OECD 210; Mingo, 2019

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.019 mg/L

Additional information

OECD 210 (2019)

The objective of the study was to assess the effects of the test item on the early-life stages of Zebrafish Danio rerio and the determination of EC10, 20, 50 values, the lowest observed effect concentration (LOEC) and hence, the no observed effect concentration (NOEC), where possible.

Materials & Methods

Test item: Amyl Cinnamic Aldehyde, batch number: 51757: (2E)-3-phenyl-2-pentyl-prop-2-enal, purity (analysed): 98.43 % w/w.

Test species: Danio rerio Hamilton (Cypriniformes: Cyprinidae).

Test design: The test was performed as a concentration-response test in a flow-through design with a duration of 35 days and renewal of stock solutions once per week, every Wednesday. Test solutions were prepared by dilution of the Test item in dimethylformamide (DMF) and application of the stock solutions into mixing chambers before being transferred to the testing chambers by means of PTFE tubes once per week.  Flow-rate was equivalent to 6 test chamber volumes per 24 hours.

For the test concentrations and the control 80 fertilised eggs (divided in four groups of 20 eggs each) were used.

Hatching and mortality of the test fish were observed daily. Records were made on mortality and visible abnormal appearance and behaviour of fish. Length, wet and dry weight of the surviving fish were measured at test end. Analytical determinations of Amyl Cinnamic Aldehyde content in test solutions were regularly conducted.

Test rates: Following a flow-through non-GLP range-finding test with nominal test item concentrations of 0.0100, 0.100 and 1.00 mg/L and a solvent control, a main test with nominal concentrations of 0.0060, 0.019, 0.060, 0.19 and 0.60 mg/L, control and solvent control was performed.

Endpoints: EC10, 20, 50, LOEC (Lowest Observed Effect Concentration) and the NOEC (No Observed Effect Concentration).

Test conditions: Temperature, pH-value and dissolved oxygen saturation of the test solutions were measured every seven days in test solutions in all test replicates (including test start and test end). Temperature was also monitored continuously in one control vessel. Water hardness was measured at the beginning of the test and at test end in vessel of each treatment. Light intensity was measured over four representative aquaria.

Samples analysed: At test start, three analytical samples taken from control and solvent control were analysed. Additionally, samples from one replicate were analysed for all test item concentrations. At days 7 and 14, 21, 28 and 35, one sample was analysed for all test item concentrations. Additionally, one sample was measured for the control and solvent control after 7, 21 and 35 days.

Statistics: All statistical calculations were carried out using the software-program ToxRat 3.3.0. ECx, NOEC and LOEC were determined by using an appropriate multiple comparison method.

Results

Validity criteria: Control survival and post-hatch success: In the control the overall survival of fertilised eggs should be greater or equal to 70 % and post-hatch success should be greater or equal to 75 %. In this study, the control survival of fertilized eggs was 97.5 % and post-hatch success was 96.2 %. The survival of fertilized eggs in the solvent control was 98.8 %, with a post-hatch success of 94.9 %.

Oxygen saturation: The dissolved oxygen concentration in control and test vessels should be at least 60 % of the air saturation value throughout the test. In this test, the dissolved oxygen concentration was ≥ 62 % % of air saturation throughout the test.

Water temperature: The water temperature should not differ by more than  1.5 °C between the test vessels or between successive days at any time during the test and should be within the temperature range of 26.0  1.5 °C. In this study, the water temperature validity criteria were fulfilled.

Test concentrations: The analytical measure of the test concentrations is compulsory.

Test conditions: The total hardness (as CaCO3) of the water was determined to be 14°dH corresponding to 250 mg/L CaCO3. The mean pH-value of the control was determined to be 7.51  0.15, the mean temperature of all treatments was measured to be 25.6  0.3 °C and the oxygen concentration was determined to be 89  11 % of the air saturation.

Analytical Results: The initial measured content of test item was between 78 and 94 % of nominal and the aged measured content at test end (day 35) was between 26 and 48 % of nominal. Since the content of test item in the samples was not between 80 and 120 % of nominal throughout the entire study, the toxicological endpoints were evaluated using arithmetic mean measured concentrations.

Statistical Results: ECx, NOEC and LOEC-values of fish early-life stages exposed to the Amyl Cinnamic Aldehyde (based on measured concentrations)

Conclusions

According to the results of the test, the overall NOEC (35 d) was determined to be 0.00384 mg/L Amyl Cinnamic Aldehyde (measured). The overall LOEC (35 days) was determined to be 0.0122 mg/L Amyl Cinnamic Aldehyde (measured). The EC10 (35 days) values of the most sensitive parameter (fresh weight) was determined to be 0.019 mg/L Amyl Cinnamic Aldehyde (measured). The EC20 (35 days) values of the most sensitive parameter (fresh weight) was determined to be 0.105 mg/L Amyl Cinnamic Aldehyde (measured). No EC50-values could be statistically determined.