rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Oct 2014 to 05 Dec 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples fall all concentrations and controls were taken from each treatment at the start of the test and at end of each renewal period. However, from the undiluted filtrate, the last samples were taken after 6 days of exposure since all test organisms in this treatment were dead at this observation date. The samples were stored deep-frozen (at about -20 °C) and protected from light immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test and before each test medium renewal by dispersing 200 mg of the test item in 2000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours in the dark at room temperature, to dissolve a maximum amount of the test item in the dispersion. After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.45 μm) after preconditioning the filter with the dispersion. The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. The test media were freshly prepared just before the introduction of eggs and larvae, respectively (at the start of the exposure and before each test medium renewal).
- Controls: Blank control (test water without test item)
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test media. All test media were clear throughout the entire test duration.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Strain: Rafinesque 1820, Teleostei, Cyprinidae

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: The spawning of the fish starts in the morning after light has been turned on. The eggs were collected from the ceiling of the spawning substrates (made of stainless steel in shape of a half-tube) as soon as possible after the eggs had been fertilised
- Subsequent handling of eggs, embryos and larvae: Embryos were preferably immersed in the test solutions before cleavage of the blastodisc or as close as possible after this stage.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Remarks on exposure duration:

The test duration included 4 days embryonic stage followed by a 4 day post hatch period based on the development in the control.

Post exposure observation period:

None reported

Hardness:

125 mg/L as CaCO3

Test temperature:

26.1 to 26.7 °C

pH:

7.2 to 7.4

Dissolved oxygen:

7.8 to 8.4 mg/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The undiluted filtrate (loading rate 100 mg/L) and 1:22, 1:10, 1:4.6 and 1:2.2 dilutions thereof, corresponding to arithmetic mean measured concentrations of 3.1, 7.0, 15, 32 and 69 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessels (8.5 cm in diameter)
- Fill volume: Approximately 200 mL test medium per vessel
- Aeration: No
- Renewal rate of test solution: 72 hour renewal period on two occasions (day 0 - 3 and day 3 - 6) and 48 hour renewal period on one occasion (day 6 - 8).
- No. of fertilized eggs per vessel: 10
- No. of vessels per concentration: 3
- No. of vessels per control: 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified water. Analytical grade salts were dissolved in the water to prepare the reconstituted test water used in the study
- Ca/mg ratio: 4:1
- Culture medium different from test medium: Reconstituted test water was used in the study and for mating of adult fish.
- Intervals of water quality measurement: The pH values, dissolved oxygen concentrations and water temperature in the test media and the control were recorded at the start and the end of each test medium renewal period as well as the appearance of the test item in test water.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16-hour light to 8-hour darkness photoperiod (with a 30 minute transition period)
- Light intensity: Approximately 240 - 290 Lux
- Other: The fish were not fed during the test and the test was stopped 4 days post-hatch before the yolk-sac of any larvae in any of the test chambers was completely absorbed and before adverse effects due to starvation started in the control.

EFFECT PARAMETERS MEASURED: The embryonic development was recorded for each replicate test chamber each day. Criteria for mortality of embryos were loss of translucency of the eggs and change in colour (caused by coagulation and/or precipitation of protein). Hatching and survival of the larvae during the hatching period were recorded every day. Mortality criteria for larvae were immobility, and lack of reaction to mechanical stimulus. Visible abnormalities like abnormal appearance (body shape) and behavior (e.g. uncoordinated swimming, atypical quiescence) of the larvae was recorded daily. The individual total length of surviving animals (tip of snout to end of tail fin) was recorded at the end of the test.

RANGE-FINDING STUDY
- Test concentrations: The undiluted filtrate (loading rate 100 mg/L) and 1:100 and 1:10 dilutions thereof.
- Results used to determine the conditions for the definitive study: Yes, in the rangefinder 17 % mortality observed after 8 days in the control and 1:100 and 1:10 dilutions and 100 % mortality in the undiluted filtrate after 5 days exposure. No deformed larvae were observed.

Reference substance (positive control):

no

Key result

Duration:

4 d

Dose descriptor:

EC10

Effect conc.:

6.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

4 d

Dose descriptor:

LOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

larval development

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

larval development

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: larvae survival; fish body length

Duration:

8 d

Dose descriptor:

LOEC

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: larvae survival; fish body length

Remarks on result:

not determinable

Remarks:

No larvae hatched at the highest test concentration of 69 mg/L

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: sublethal effects

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: sublethal effects

Duration:

4 d

Dose descriptor:

EC50

Effect conc.:

> 32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: embryo development rate, larvae survival, fish length

Details on results:

- Mortality/survival at embryo and larval stages: The mean survival was 88 % in the control. The mean survival success at the test concentrations of 3.1 to 32 mg/L were nearly identical or even slightly higher compared to the control and no concentration-effect relationship was observed. At the highest mean measured concentration of 69 mg/L, only one larvae hatched at day 4 post fertilization but died 24 hours later. Therefore, this test concentration was not considered for the evaluation of the NOEC of the survival success.
- Days to hatch and numbers hatched: At Day 4 first hatching was observed at all test concentrations and the control. Day 4 was also determined to be the end of the hatching period as within this day more than 90 % of the viable embryos had hatched in the control and the test concentrations of 3.1 and 7.0 mg/L (both test concentrations without observed toxic effect). At the higher test concentrations of 15 and 32 mg/L end of hatching was delayed by one day which was attributed to an adverse effect of the test item. At the mean measured concentration of 69 mg/L, only one hatched larvae was recorded at Day 4, but this larvae had died at Day 5 and thus was regarded as not successfully hatched.
- Data for length and weight of surviving fish: For the fish body length the mean values obtained from all test concentrations up to and including 32 mg/L were not statistically different to the control.
- Type of and number with behavioural abnormalities: All larvae which survived until the end of the test at the concentrations of 3.1 to 15 mg/L were healthy and showed normal behavior. At the mean measured concentration of 32 mg/L, all surviving larvae showed apathy after hatching until test end which was regarded as a sublethal effect.
- Other biological observations: The development rate of the embryos from fertilization to Day 5 (end of hatching at all test concentrations) was found to be statistically significant impacted first at the test concentration of 32 mg/L (Welch t-test, one-sided smaller, α = 0.05). At the highest mean measured concentration of 69 mg/L, only one larvae hatched at day 4 post fertilization but died 24 hours later and thus was regarded as not successfully hatched.
- Incidents in the course of the test which might have influenced the results: The test item proved to be stable in test water during the renewal periods.

Reported statistics and error estimates:

The 4-day EC10 and 4-day EC50 for hatching success was calculated by Probit Analysis. The EC50 for length, survival success and development rate could not be calculated due to the absence of a toxic effect up to the highest concentration with surviving larvae or due to the low toxic effect observed. The overall NOEC (based on hatching success and development rate) was reported. The highest test concentration of 69 mg/L was not taken into account for the estimation of the EC50 nor for the NOEC calculation for embryo development.

Table 1. Summary of results

Mean measured concentration (mg/L)	Hatching success#		Development rate of viable embryos		Survival success		Body length of surviving fish at the end of the test
	Mean hatching success at Day 4 (%)	% of control at Day 4	Mean ± SD (1/day)	% of control at Day 5	Mean ± SD (%)	% of control	Range (mm)	Mean ± SD (mm)	% of control
Control	80&	-	0.283 ± 0.0041&	-	88 ± 12.5	-	4.7 - 5.9	5.4 ± 0.3	-
3.1	80	100	0.283 ± 0.0039	100	100 ± 0.0	114	4.7 - 6.3	5.4 ± 0.4	99.8
7.0	73	92	0.283 ± 0.0046	100	96 ± 7.2	109	4.6 - 6.0	5.5 ± 0.3	101.3
15	37	46*	0.256 ± 0.0293	90	100 ± 0.0	114	4.5 - 6.1	5.4 ± 0.4	99.3
32	17	21*	0.240 ± 0.0075	85*	85 ± 14.4	97	4.7 - 5.7	5.3 ± 0.0	98.1
69	3	4*	n.a.	n.a.	0	0	n.a.	n.a.	n.a.

* Statistically significantly reduced compared to control (Fishers Exact Binominal Test, one sided greater, α = 0.05 for hatching success, larvae survival or Welch t-test, one-sided smaller, α = 0.05 for embryo development rate or one-way ANOVA, p < 0.05 for body length)

# 10 eggs per replicate were inserted at test start. Dead eggs were not included in the evaluation of hatching success. Day 4 was determined to be the end of the hatching period as more than 90 % of the viable embryos have hatched in the control.

SD Standard deviation

n.a. The highest test concentration of 69 mg/L was not considered for the evaluation of the NOEC of development rate or for statistical analysis of body length, as only one larvae hatched on Day 4 and was found to be dead on Day 5.

& Two larvae were dead at Day 5; these were taken into consideration for the calculation of the development rate.

Validity criteria fulfilled:

yes

Remarks:

The hatching success in the control was 80% (must be ≥60%) and survival of the hatched larvae was 88% (must be ≥70%). The dissolved oxygen concentration was ≥96% (must be 60-100%) and the water temperature was 25±2.0°C and did not differ by > ±1.5°C.

Conclusions:

The most sensitive endpoint for Pimephales promelas was determined to be hatching success, with an 4-day NOEC of 7.0 mg/L, an 4-day LOEC of 15 mg/L, a 4-day EC10 of 6.2 mg/L and a 4 -day EC50 of 16 mg/L. For embryo development rate and sublethal effects, the NOEC was 15 mg/L and the LOEC was 32 mg/L. For larvae survival and fish length, the NOEC was 32 mg/L and the LOEC could not be determined as no larvae had survived at the test concentration of 69 mg/L. The 4-day EC50 for embryo development rate, larvae survival and fish length was > 32 mg/L.

Executive summary:

The toxicity of the test item to fathead minnow (Pimephales promelas) was determined in an 8-day semi-static test with test medium renewal at Day 3 and Day 6, according to the OECD Guideline for Testing of Chemicals No. 212 (1998). Due to the low water solubility of the test item, a dispersion of the test item with a nominal loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 3 hours and filtered before use. The undiluted filtrate and dilutions of 1:2.2, 1:4.6, 1:10 and 1:22 were used as test media, corresponding to arithmetic mean measured concentrations of 3.1, 7.0, 15, 32, and 69 mg/L. Additionally, a control was tested in parallel. The most sensitive endpoint was determined to be hatching success, with an 4-day NOEC of 7.0 mg/L, an 4-day LOEC of 15 mg/L, a 4-day EC10 of 6.2 mg/L and a 4 -day EC50 of 16 mg/L. For embryo development rate and sublethal effects, the NOEC was 15 mg/L and the LOEC was 32 mg/L. For larvae survival and fish length, the NOEC was 32 mg/L and the LOEC could not be determined as no larvae had survived at the test concentration of 69 mg/L. The 4-day EC50 for embryo development rate, larvae survival and fish length was > 32 mg/L.

Description of key information

4 -d NOEC = 6.2 mg/L, freshwater fathead minnow (Pimephales promelas), OECD 212, Eckenstein 2015

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

6.2 mg/L

Additional information

The toxicity of the test item to fathead minnow (Pimephales promelas) was determined in an 8-day semi-static test with test medium renewal at Day 3 and Day 6, according to the OECD Guideline for Testing of Chemicals No. 212 (1998). Due to the low water solubility of the test item, a dispersion of the test item with a nominal loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 3 hours and filtered before use. The undiluted filtrate and dilutions of 1:2.2, 1:4.6, 1:10 and 1:22 were used as test media, corresponding to arithmetic mean measured concentrations of 3.1, 7.0, 15, 32, and 69 mg/L. Additionally, a control was tested in parallel. The most sensitive endpoint was determined to be hatching success, with an 4-day NOEC of 7.0 mg/L, an 4-day LOEC of 15 mg/L, a 4-day EC10 of 6.2 mg/L and a 4 -day EC50 of 16 mg/L. For embryo development rate and sublethal effects, the NOEC was 15 mg/L and the LOEC was 32 mg/L. For larvae survival and fish length, the NOEC was 32 mg/L and the LOEC could not be determined as no larvae had survived at the test concentration of 69 mg/L. The 4-day EC50 for embryo development rate, larvae survival and fish length was > 32 mg/L.