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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 March 2015 to 22 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

yes

Remarks:

The difference in temperature range was 2.92 °C, exceeding the required range of ± 1 °C. As the temperatures were within the recommended range (18 - 22 °C) and the control validity criterion was met, the quality and integrity of the study is not affected.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All treatment groups and the control
- Sampling method: Three samples were collected from each treatment group and one from the control group in the freshly prepared media on 0, 2, 12, and 14 days after exposure and in the expired media on 2, 12, 14 and 21 days after exposure.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate weights of the test item were dissolved in 25 mL acetone. 5 mL of the acetone solution was applied into dry glass bottles and the bottles were rotated to allow the acetone to evaporate, thus leaving a thin coating of test item on the inner surface. Afterwards, 5 L of test water was added into the glass bottles and stirred magnetically for 1 to 1.5 hrs (400 to 500 rpm) to obtain a stock solution of 10 mg/L. Appropriate volumes of the stock solution were diluted with test water to 2 L and stirred magnetically for 20 to 30 minutes (400 to 500 rpm).
- Controls: Blank control
- Concentration of vehicle in test medium: Acetone was evaporated from the test vessel before introduction of test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Age of parental stock: Test animals were derived from a healthy pre-cultured population (i.e. showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc) and were less than 24 hours old and not first brood progeny.
- Feeding during test: Yes
- Food type: Living algal cells of Chlorella sp and Desmodesmus subspicatus
- Amount: Concentrated algal suspension, providing between 0.1 and 0.2 mg organic carbon/Daphnia/day
- Frequency: Daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

None reported

Hardness:

142 mg CaCO3/L

Test temperature:

19.00 - 21.92 °C

pH:

7.28 - 8.53

Dissolved oxygen:

8.26 - 10.09 mg/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal test item concentrations: 0.20, 0.53, 1.4, 3.8 and 10 mg/L
Mean measured test item concentrations 0.192, 0.526, 1.397, 3.819 and 10.198 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Type: Closed; loosely covered to reduce the loss of water due to evaporation and to avoid the entry of dust
- Material, size, headspace, fill volume: 250 mL beakers, filled with approximately 100 mL test solution
- Aeration: Water was aerated for at least 24 hours to attain dissolved oxygen saturation before use
- Renewal rate of test solution: 3 times per week, on Monday, Wednesday and Friday
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: Approximately 100 mL/animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater treated with Aquapro Pure Water System
- Total organic carbon: 1.74 mg/L
- Metals: As < 0.001 mg/L, Cd < 0.001 mg/L, Cr(6+) < 0.004 mg/L, Pb < 0.005 mg/L, Hg < 0.0001 mg/L, Al < 0.03 mg/L, Fe < 0.05 mg/L, Cu < 0.0075 mg/L, Zn < 0.05 mg/L
- Culture medium different from test medium: Culture conditions (light, temperature and medium) were similar to those used in the test.
- Intervals of water quality measurement: The hardness and TOC of the test water was measured at the beginning of the test. The pH and dissolved oxygen in fresh and expired test media was determined once or twice a week. The light intensity was determined at the beginning and end of the test. Temperature was recorded in 1 hour intervals.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16 hours light: 8 hours dark
- Light intensity: Cool white light, 1008 - 1451 lux

EFFECT PARAMETERS MEASURED: The number of living offspring was recorded daily from the appearance of the first brood. Parental mortality and abnormal symptoms were recorded daily. Body length (excluding the anal spine) in parental animals was measured at the end of the test. The time to production of first brood and the total broods of each parent were also recorded.

TEST CONCENTRATIONS
- Range finding study: A non-GLP range-finding test was conducted with nominal concentrations of 0.1, 0.4, 1, 5, 10 mg/L, concurrently with a blank control group., 0.53654g, 0.72410g, 0.97786g and 1.32147g test item.
- Test concentrations: 0.1, 0.4, 1, 5 and 10 mg/L
- Results used to determine the conditions for the definitive study: At 0.1, 0.4 and 1.0 mg/L, the number of juveniles from the parent animals were not significantly different compared to the control. At 10 mg/L, the number of juveniles from the parent animals were significantly different compared to the control. Reproduction could not be assessed in the 5 mg/L treatment group as all parental animals died. As all parent animals were alive at the higher concentration of 10 mg/L, this is considered to be due to accidental death or other unknown reasons.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.526 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: total living offspring for each parental animal

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.397 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: total living offspring for each parental animal

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.397 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

body length of parental animal

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.819 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

body length of parental animal

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.819 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental animal

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

10.189 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental animal

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.819 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: time to production of first brood

Duration:

21 d

Dose descriptor:

LOEC

Basis for effect:

other: time to production of first brood

Remarks on result:

not determinable

Remarks:

all test organisms in measured concentration 10.189 mg/L were dead before pregnancy

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.819 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: total broods of each parent

Duration:

21 d

Dose descriptor:

LOEC

Basis for effect:

other: total broods of each parent

Remarks on result:

not determinable

Remarks:

all test organisms in measured concentration 10.189 mg/L were dead before pregnancy

Details on results:

- Behavioural abnormalities: All test organisms in measured concentration 10.189 mg/L were lethargic or immobilised beginning on day 2 of exposure until death.
- Observations on body length and weight: Body length displayed significant differences (p < 0.05) between the control and the measured concentration of 3.819 mg/L.
- Other biological observations: At the measured concentration of 3.819 mg/L, two parent animals died accidentally or inadvertently, so the two replicates were excluded from the analysis. The mortalities were considered as accidental or inadvertent as the animals did not show any abnormal symptoms before death. Parental animal mortality displayed significant differences (p < 0.05) between the control and the measured concentration of 10.189 mg/L. The test organisms in measured concentration 10.189 mg/L all died before pregnancy at 4 or 5 days of exposure.
- Mortality of control: There was no mortality of parent animals in the control.
- Any observations that might cause a difference between measured and nominal values: The mean measured concentrations in the expired test solutions were in the range of 82.9 to 98.9 % of the initial measured concentrations. Mean measured concentrations in the test media were in the range of 95.8 to 101.9 % of the nominal concentrations.

Reported statistics and error estimates:

The NOEC and LOEC were determined by two-sided Student T-test at 5 % significance level. Replicates where the parent animals were considered to have died accidentally or inadvertently during the test were excluded from the analysis.

Table 1. Summary of results

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Total living offspring of each parent animal		Body length of each parent animal (mm)		Mortality of parent animals (%)		Time to production of first brood (days)		Total broods of each parent
		Mean ± SD	p value	Mean ± SD	p value	Mean ± SD	p value	Mean ± SD	p value	Mean ± SD	p value
Control	nd	212 ± 14	-	3.51 ± 0.20	-	0 ± 0.0	-	7.2 ± 0.63	-	5.8 ± 0.4	-
0.20	0.192	215 ± 16	0.703	3.39 ± 0.20	0.14	0 ± 0.0	1.0	7.7 ± 1.06	0.096	5.8 ± 0.4	1.00
0.53	0.526	209 ± 27	0.586	3.42 ± 0.21	0.52	0 ± 0.0	1.0	7.4 ± 0.97	0.619	5.8 ± 0.7	1.00
1.4	1.397	172 ± 17*	0.011	3.35 ± 0.22	0.058	0 ± 0.0	1.0	7.4 ± 0.70	0.343	6.0 ± 0.9	0.443
3.8	3.819	132 ± 13**#	0.003	3.02 ± 0.14***#	1.91E-04	0 ± 0.0	1.0	7.9 ± 0.88	0.066	5.5 ± 0.5#	0.170
10	10.189	na	na	na	na	100 ± 0.0***	0.0	na	na	na	na

* Significantly different compared to the control, p < 0.05

** Significantly different compared to the control, p < 0.005

*** Significantly different compared to the control, p < 0.001

SD: Standard deviation

nd: Not determined; the signal was lower than 30 % of signal of the 0.1 mg/L fortified concentration

na: Not assessed; the test organisms in measured concentration 10.189 mg/L all died before pregnancy at 4 or 5 days of exposure

#: Two replicates died accidentally or inadvertently so the replicates were excluded from the analysis

Validity criteria fulfilled:

yes

Remarks:

In the control, mortality was < 20 %, mean number of living offspring was ≥ 60, coefficient of variation of the number of living offspring was < 25 %. In one of the treatment groups, ≤ 20 % of test animals died accidentally or inadvertently.

Conclusions:

In a 21-day reproduction test with Daphnia magna, the most sensitive endpoint was determined to be total living offspring produced per parent animal, with a NOEC of 0.526 mg/L and LOEC of 1.397 mg/L. For the remaining parameters, the NOEC values were 1.397 mg/L based on parent body length and 3.819 mg/L based on parent mortality, time to production of first brood and total broods per parent. The LOEC values were 3.819 mg/L based on parent body length and 10.189 mg/L based on parent mortality. The LOEC values for time to production of first brood and total broods per parent could not be determined as all test organisms died before pregnancy at the highest test concentration.

Executive summary:

A 21-day reproduction test with the test material was conducted with Daphnia magna in a semi-static test system according to OECD Guideline Number 211 (2012). Test animals were exposed to nominal test item concentrations of 0.20, 0.53, 1.4, 3.8 and 10 mg/L (corresponding to mean measured test item concentrations of 0.192, 0.526, 1.397, 3.819 and 10.189 mg/L), alongside a blank control. Test solutions were renewed three times per week (Monday, Wednesday and Friday). The most sensitive endpoint was determined to be total living offspring produced per parent animal, with a NOEC of 0.526 mg/L and LOEC of 1.397 mg/L. For the remaining parameters, the NOEC values were 1.397 mg/L based on parent body length and 3.819 mg/L based on parent mortality, time to production of first brood and total broods per parent. The LOEC values were 3.819 mg/L based on parent body length and 10.189 mg/L based on parent mortality. The LOEC values for time to production of first brood and total broods per parent could not be determined as all test organisms died before pregnancy at the highest test concentration. All test organisms in measured concentration 10.189 mg/L were lethargic and immobilised beginning on day 2 of exposure until death.

Description of key information

21-d NOEC = 0.526 mg/L, freshwater (Daphnia magna), OECD 211, Xue 2015.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.526 mg/L

Additional information

A 21-day reproduction test with the test material was conducted with Daphnia magna in a semi-static test system according to OECD Guideline Number 211 (2012). Test animals were exposed to nominal test item concentrations of 0.20, 0.53, 1.4, 3.8 and 10 mg/L (corresponding to mean measured test item concentrations of 0.192, 0.526, 1.397, 3.819 and 10.189 mg/L), alongside a blank control. Test solutions were renewed three times per week (Monday, Wednesday and Friday). The most sensitive endpoint was determined to be total living offspring produced per parent animal, with a NOEC of 0.526 mg/L and LOEC of 1.397 mg/L. For the remaining parameters, the NOEC values were 1.397 mg/L based on parent body length and 3.819 mg/L based on parent mortality, time to production of first brood and total broods per parent. The LOEC values were 3.819 mg/L based on parent body length and 10.189 mg/L based on parent mortality. The LOEC values for time to production of first brood and total broods per parent could not be determined as all test organisms died before pregnancy at the highest test concentration. All test organisms in measured concentration 10.189 mg/L were lethargic and immobilised beginning on day 2 of exposure until death.