Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

26 Jun 2007 to 28 Aug 2007

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:

September 21, 1998

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals and methods: For the determination of the test item concentrations, duplicate samples were taken from the freshly prepared test media of all test concentrations and the solvent control at the first treatment period (Day 0), at a treatment period in the second week (Day 7), and at a treatment period in the last week (day 16). To determine the maintenance of the test item concentrations during the test medium renewal periods, duplicate samples were taken from all test media and the solvent control at the end of two test medium renewal periods of 48 (on Days 2 and 9) and of one test medium renewal period of 72 hours (Day 19). The samples were taken from the actual test by combining the contents of the test beakers after the end of the treatment period.
- Storage: All samples were deep-frozen (at about -20°C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (without GLP), the isomers of the test item were considered to be stable in the test water under these storage conditions.

Vehicle:

yes

Remarks:

N,N-Dimethylformamide (DMF)

Details on test solutions:

The application solution for the dosage of the highest test concentration was prepared by completely dissolving 32.5 mg of the test item (effective weights in the range of 32.5 to 32.6 mg) in 25 mL of DMF by intense mixing. Further dilutions of this application solution with DMF were performed to obtain the application solutions for the dosage of the lower test concentrations.
The application solutions were freshly prepared once a week (three preparations during the test period) and were used for all test media preparations during this week. Between the test medium renewal periods, the application solutions were stored in a refrigerator at approximately 4 ˚C. The stability of the application solutions during their use was examined in a pre-experiment (without GLP) and was confirmed within this study.
For the preparation of the test media, adequate volumes of the application solutions were added to the test water (100 µg/L per litre test water) during intense stirring to obtain the test concentrations as stated above. Then, the test media were stirred for 15 minutes to ensure that the test item was completely dissolved. The concentration of DMF was the same in all test media and in the solvent control (100 µg/L DMF per litre test water). For the control, test water without addition of test item or solvent was used.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water fleas
- Strain/clone: Defined by the supplier as clone 5; the clone is successfully bred in the test facility's laboratories in culture medium identical to the medium used for the test.
- Age of the organism (at the start of the test): < 24 hours old (The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny).
- Feeding during test : The test animals were fed with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (grown in the laboratories of the test facility) and a fish food suspension. The fish food suspension was fed in addition to the algal food because a toxic effect of the test item on algae could not be excluded.
- Feeding amount: The carbon contents of the algal and fish food suspensions were determined. The food amounts were based on the measured
concentrations of total organic carbon (TOC) in the food suspensions and consisted of 50% algae and 50% fish food (based on TOC).
The amounts of TOC fed per test animal and day were as follows:
Day 0/1: 0.10 mg TOC / Daphnia
Day 2/5-8/12-13: 0.15 mg TOC / Daphnia
Day 14-15/19-20: 0.20 mg TOC / Daphnia
Day 9/16: 0.25 mg TOC / Daphnia
On Day 9 and 16 (Fridays), the amount of food was increased to 0.25 TOC per Daphnia to provide a sufficient amount of food for the following two days (Saturday and Sunday).
- Frequency: On each working day (Monday through Friday)

CULTIVATION
- Culture condition: The stock animals were maintained in one liter glass beakers filled with culture medium and were transferred twice a week to fresh medium.
- Type and amount of food: The stock animals were fed with green algae of the species Scenedesmus subspicatus and with a fish food suspension.
- Feeding frequency: The animals were fed normally three times a week.
- Health during Cultivation: The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.5 mmol/L (250 mg/L as CaCO3)

Test temperature:

20 - 21 °C

pH:

7.6 - 8.2

Dissolved oxygen:

8.0 - 9.1 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (solvent control), 0 (negative control), 0.40, 1.3, 4.0, 13, 40 and 130 µg/L
As mean measured concentrations ranged between 84 and 88%, nominal concentrations were used for the calculation and reporting of the results.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers containing 80 mL medium and covered with glass plates
- Type: Close (covered with glass plates)
- Aeration: None
- Renewal rate of test solution (frequency): This semi-static test with test medium renewals every 48 hours or 72 hours.
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10
- No. of vessels per vehicle control: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test was conducted in reconstituted water with an initial pH of 7.9. Before use, the test water was aerated until oxygen saturation. During the test, the test media were not aerated.
- Test medium: Elendt M7
- Intervals of water quality measurement: At the beginning and end of each test medium renewal period, the pH and dissolved oxygen concentrations were measured in one replicate of each test concentration and the controls. At the same time, the water temperature was measured in one of the control replicates. Additionally, the room temperature was continuously monitored. The appearance of the test media was visually recorded at the beginning and end of each test medium renewal period.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness, with a 30-minute transition period
- Light intensity: About 490 - 650 Lux

EFFECT PARAMETERS MEASURED
The test replicates were observed for mortality of adults at the start of the test, on the first and second day after the start of the study and thereafter three times per week before renewal of test media. On the observation dates, the number of offspring was recorded. The test beakers were also checked for the presence of aborted eggs or dead offspring. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test. The body length of the adults was measured at the end of the test by measuring the daphnids from the top of the head to the basis of the spina with the use of a binocular microscope.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

13 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

53 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

An overview of the results is provided in Table 1 - Table 5 in 'Any other information on results incl. tables'
- Survival: In the control, the solvent control and at the lower test item concentrations up to and including 40 µg/L, the survival of the test animals at the end of the test was at least 90% or higher. At the highest test item concentration of 130 µg/L, the survival at the end of the test was 30%. Thus, the survival of Daphnia magna after 21 days was significantly reduced (> 20%) at the highest test item concentration of 130 µg/L.
- Reproduction: The first young offspring released from their parent animals were recorded in the controls and at the lower test item concentrations up to and including 13 µg/L at the observation on Day 7. Thus, the time of the first brood was not affected up to and including this test concentration. At the next higher test item concentration of 40 µg/L, a delay of the first brood was observed (the first offspring was observed on Day 12 of exposure). At the highest test item concentration of 130 µg/L, no offspring were produced by the three daphnids surviving until the end of the test.
- Reproduction rate: The mean reproduction rate of the daphnids in the solvent control was 82.8 ± 9.5 living offspring per adult. The corresponding value in the control was 86.0 ± 7.3. According to the results of the Williams’test (one-sided smaller, α = 0.05), no inhibitory effect of the test item on the mean reproduction rate was determined up to and including the test item concentration of 13 µg/L. At the test item concentration of 40 µg/L, the mean reproduction rate of surviving daphnids was statistically significantly reduced to 83% living offspring compared to the solvent control. At the highest test item concentration of 130µg/L, reproduction of the surviving daphnids was completely inhibited. The 21-day EC50 for the inhibition of the reproduction rate of Daphnia magna (on the nominal concentrations of the test item) was calculated to be 53 µg/L (95% confidence interval: 51 - 55 µg/L).
- Body length: The mean body lengths of the daphnids in the control and the solvent control were 4.12 ± 0.14 mm and 4.06 ± 0.06 mm, respectively. Based on a Williams' test (one sided smaller, α = 0.05), the mean body length was not statistically significantly smaller compared to the solvent control at any test item concentration up to and including 40 µg/L. At the highest test item concentration of 130 µg/L, the mean body length of the three surviving daphnids was statistically significantly reduced to 84% compared to the solvent control.
Besides the effects mentioned above, dead offspring were found on Day 16 at the test concentration of 40 µg/L.

Reported statistics and error estimates:

The NOEC and the LOEC of the reproduction rate were statistically evaluated by testing the mean reproduction rate and the mean body length at the test concentrations for statistically significant differences to the solvent control value by the multiple Williams’ test.
The EC50 for the inhibition of the reproduction rate after 21 days was calculated by Probit Analysis.

Table 1: Number of surviving test animals

Exposure day	Nominal concentration of the test item (µg/L)
	Solvent control	Control	0.40	1.3	4.0	13	40	130
0	10	10	10	10	10	10	10	10
1	10	10	10	10	10	10	10	9
2	10	10	10	10	10	10	10	5
5	10	10	10	10	10	10	10	3
7	10	10	10	10	9	10	10	3
9	10	10	10	10	9	10	10	3
12	10	10	10	10	9	10	10	3
14	10	10	10	10	9	10	10	3
16	10	10	10	10	9	10	10	3
19	10	10	10	10	9	10	10	3
21	10	10	10	10	9	10	10	3
% surviving on day 21	100	100	100	100	90	100	100	30

Table 2. The total number of living young daphnids produced by all adults (cumulative values)

Exposure day	Nominal concentration of the test item (µg/L)
	Solvent control	Control	0.40	1.3	4.0	13	40	130
0	0	0	0	0	0	0	0	0
1	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	o	0
7	44	34	53	71	49	38	0	o
9	136	114	88	119	105	128	0	0
12	347	330	297	320	295	345	215	0
14	562	581	530	604	510	594	344	0
16	651	587	595	615	545	637	360*	0
19	739	634	684	669	616	727	513	0
21	828	860	873	866	784	900	687	0
% of solvent control 1	100.0	103.9	105.4	104.6	94.7	108.7	83.0	0

1. Based on the value of the last exposure day

* In addition 51 dead juveniles were found, which were not included in the evaluation.

Table 3. The number of living offspring produced per surviving adult after 21 days of exposure

	Nominal concentration of the test item (µg/L)
Replicate	Solvent control	Control	0.40	1.3	4.0	13	40	130
1	96	79	90	87	82	92	89	*
2	83	77	89	83	86	91	73	*
3	84	86	97	71	87	83	47 (13)**	*
4	88	88	91	83	*	90	72	*
5	99	90	87	84	94	115	45	*
6	75	81	85	81	100	71	88 (20)**	*
7	71	77	80	89	78	101	52	*
8	71	94	74	102	67	87	76	0
9	80	90	93	96	92	83	60	0
10	81	98	87	90	98	87	85	0
Mean	82.8	86.0	87.3	86.6	87.1	90.0	68.7	0.0
±SD	9.5	7.3	6.5	8.5	10.4	11.7	16.7	0.0
N	10	10	10	10	9	10	10	3
CV %	11.5	8.5	7.5	9.8	12.0	13.0	24.4	0.0
% of solvent control	100.0	103.9	105.4	104.6	105.2	108.7	83.0	0.0
STAT	-	-	n.s.	n.s.	n.s.	n.s.	s.	s.

SD: standard deviation

CV %: coefficient of variation in %

*: test animal died during the test period of dead offspring (not included in the evaluation)

STAT: results of a Williams’ test with the mean values of living offspring (one-sided smaller,α= 0.05)

n.s.: mean value not significantly lower than in the solvent control

s.: mean value significantly lower than in the solvent control

  Table 4. Body length [mm] of the surviving adults after 21 clays of exposure

	Nominal concentration of the test item (µg/L)
Replicate	Solvent control	Control	0.40	1.3	4.0	13	40	130
1	4.1	4.4	4.1	4.1	4.0	4.0	4.1	*
2	4.1	4.1	4.6	4.1	3.9	4.2	4.1	*
3	4.1	4.0	3.9	4.0	4.1	4.0	3.9	*
4	4.0	3.9	4.1	4.1	*	4.0	4.0	*
5	4.1	4.2	4.0	4.0	4.2	4.3	3.9	*
6	4.0	4.2	4.1	3.9	4.2	4.1	3.9	*
7	4.2	4.0	4.4	4.3	4.1	4.3	4.2	*
8	4.1	4.1	4.0	4.2	4.1	4.1	3.8	3.1
9	4.0	4.2	4.3	4.1	4.2	4.1	3.9	3.6
10	4.0	4.1	4.2	4.1	4.3	4.2	4.3	3.4
mean		4.12	4.17	4.08	4.10	4.12	4.01	3.39
±SD	0.06	0.14	0.22	0.11	0.12	0.12	0.16	0.27
n	10	10	10	10	9	10	10	3
CV %	1.5	3.5	5.2	2.7	2.9	2.9	4.0	7.8
% of solvent control	100.0	101.6	102.8	100.6	101.1	101.6	98.8	83.6
STAT	-	-	n.s.	n.s.	n.s.	n.s.	n.s.	s.

SD: standard deviation

CV %: coefficient of variation in %

*: test animal died during the test period of dead offspring (not included in the evaluation)

STAT: results of a Williams’ test with the mean values of living offspring (one-sided smaller,α= 0.05)

n.s.: mean value not significantly lower than in the solvent control

Table 5. Summary of effects of the test substance on Daphnia magna over 21 days of exposure

	Nominal concentration of the test item (µg/L)
	Solvent control	Control	0.40	1.3	4.0	13	40	130
Mortality after 21 days of exposure (%)	0	0	0	0	0	0	0	70
Mean reproduction rate (living offspring per surviving adult)	82.8	86.0	87.3	86.6	87.1	90.0	68.7 *	0.0*
Mean reproduction rate in % of solvent control	100.0	103.9	105.4	104.6	105.2	108.7	83.0	0.0
Mean body length of the adults (mm)	4.06	4.12	4.17	4.08	4.10	4.12	4.01	3.39 *
Mean body length of the adults as % of solvent control	100.0	101.6	102.8	100.6	101.1	101.6	98.8	83.6

* statistically significantly lower than value in the solvent control (results of a Williams’ test, one-sided,α= 0.05)

s.: mean value significantly lower than in the solvent control

Validity criteria fulfilled:

yes

Conclusions:

In a reproduction study on Daphnia magna, performed in accordanc with OECD TG 211, the 21-day NOEC for reproduction was determined to be 13 µg/L, based on nominal concentrations.

Executive summary:

The impact of the test substance on the reproduction of Daphnia magna was studied in a semi-static system for 21 days. The study was conducted in accordance with OECD TG 211 and in compliance with CLP criteria. The test organisms (first instar < 24-hours old) were exposed to the test substance in reconstituted test water at 20 ± 1 ˚C and 16 hours light and 8 hours darkness, with a 30-minute transition period (approx. 490 - 650 Lux). The organisms (1 daphnia/vessel; 10 vessels/concentration or control) were exposed to 0 (N,N-Dimethylformamide; solvent control), 0 (negative control), 0.40, 1.3, 4.0, 13, 40 and 130 µg/L of the test substance, based on nominal concentration. The test replicates were observed for mortality of adults at the start of the test, on the first and second day after the start of the study and thereafter three times per week before renewal of test media. On the observation dates, the number of offspring was recorded. The test beakers were also checked for the presence of aborted eggs or dead offspring. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test. The body length of the adults was measured at the end of the test by measuring the daphnids from the top of the head to the basis of the spina with the use of a binocular microscope.

The measured concentration of syn-isomer in the old and new test media were between 75 and 95% of the nominal values. The measured concentration of anti-isomer in the old and new test media were between 75 and 92% of the nominal values. Therefore, the test item was stable in the test medium over the renewal periods of 48 and 72 hours. As mean measured concentrations ranged between 84 and 88% nominal concentrations were used for the calculation and reporting of the results. Survival of the parental generation animals was 100% in the solvent control and in the control, and in all test concentrations up to and including 1.3 μg/L. One parental generation animal (10%) died by day 7 at 4.0 μg/L. There were no parental generation mortalities at 13 or 40 μg/L, while at the highest concentration (130 μg/L) 70% of the parental generation had died by day 5. The remaining three animals survived to the end of the test, but did not reproduce.The first brood juveniles were observed on day 7 in the controls and all test concentrations up to and including 13 μg/L. Hence, the time to first brood was unaffected at these concentrations. At 40 μg/L, the first brood animals were not seen until day 12, while no juveniles were produced throughout the test at 130 μg/L.The mean number of juveniles per surviving adult showed a statistically significant inhibitory effect on the reproduction of D. magna over 21 days at concentrations of 40 μg/L and above (Williams-test, p=0.05). Based on the findings, the 21-day NOEC for reproduction was determined to be 13 µg/L, based on nominal concentrations.

Description of key information

Freshwater, 21 -d NOEC = 13 µg/L (based on nominal concentrations), reproduction, Daphnia magna, OECD TG 211, Bätscher 2007

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

13 µg/L

Additional information

One study is available for this endpoint, which was selected as key study. In this study performed according to OECD TG 211 and in compliance with GLP, Daphnia magna (first instar < 24-hours old) were exposed to the test substance at concentrations of 0 (N,N-Dimethylformamide; solvent control), 0 (negative control), 0.40, 1.3, 4.0, 13, 40 and 130 µg/L of the test substance. The test organisms (1 daphnia/vessel; 10 vessels/concentration or control) were exposed to the test substance in reconstituted test water at 20 ± 1 ˚C and 16 hours light and 8 hours darkness, with a 30-minute transition period (approx. 490 - 650 Lux) for 21 days.

After 21 days exposure, survival of the parental generation animals was 100% in the solvent control and in the control, and in all test concentrations up to and including 1.3 μg/L. One parental generation animal (10%) died by day 7 at 4.0 μg/L. There were no parental generation mortalities at 13 or 40 μg/L, while at the highest concentration (130 μg/L) 70% of the parental generation had died by day 5. The remaining three animals survived to the end of the test, but did not reproduce. The first brood juveniles were observed on day 7 in the controls and all test concentrations up to and including 13 μg/L. Hence, the time to first brood was unaffected at these concentrations. At 40 μg/L, the first brood animals were not seen until day 12, while no juveniles were produced throughout the test at 130 μg/L. The mean number of juveniles per surviving adult showed a statistically significant inhibitory effect on the reproduction of D. magna over 21 days at concentrations of 40 μg/L and above. Based on the findings, the 21-day NOEC for reproduction was determined to be 13 µg/L (nominal).