3-methoxy-N,N-dimethylpropionamide

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:

supporting study

Study period:

27 April 2012 - 18 May 2012

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:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples analysis were taken from each test solution and the control (on one replicate vessel).
Frequency: in fresh media on day 0, day 12 and day 19; in old media on day 3, day 14 and day 21.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of 80 mg test substance was diluted in 2000 mL Elendt M4 media (stock solution concentration: 40.0 mg/L). The test solutions were prepared by appropriate dilution of this stock solution. The test solutions used in the test were prepared without the use of a solubilising agent, by mechanical dispersion. Test solutions were renewed three times a week, based on the stability of the test substance.
- Controls: test medium without test item or other additives.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus
- Source: National Institute of Public Health 1097 Budapest Gyali u. 2-6 Hungary. Culture used for the test do not contain ephippia, no mortality was observed within the stock culture 2 days before the start of the test and daphnids were not used in any portion of a previous test.
- Age at study initiation: <24 hours
- Feeding during test: once per day with concentrated algal suspension of Pseudokirclmeriella subcapitata, corresponding to 0.15 mg C/Daphnia/day.
- Number of animals: 10 per concentration and for the control, parent animals were maintained individually, one per test vessels with 80 mL of test solution in each vessel.

BREEDING:
- Breeding conditions: in Ecotoxicological Laboratory of CiToxLAB Hungary Ltd., in the same medium as the test.
- Acclimation: To avoid the necessity of adaptation prior to the test the quality of water used was similar to the culture media.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

> 140 mg CaCO3/L

Test temperature:

20.5 - 21.1 °C throughout the test

pH:

7.6 - 8.0 throughout the test

Dissolved oxygen:

8.0-8.8 mg/L

Nominal and measured concentrations:

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test:
Nominal concentrations: 2.5, 5.0, 10, 20 and 40 mg/L
Measured concentrations: for detailed measured concentrations see table 1. Measured concentrations ranged from 94-103% of nominal and in one case 117% of nominal. Based on these results, the final effect concentrations were based on nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel fill volume: ca. 80 mL
- Aeration: no
- Renewal rate of test solution: 2-3 days; When the medium was renewed a second series of test vessels were prepared and the parent animals transferred to them by a pipette of suitable diameter. The volume of
test medium transferred with the Daphnia was minimised.
- No. of organisms per vessel: 1 neonate daphnid
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 media
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, oxygen concentration and pH were measured once a week in fresh and old media, in the control and in the highest test item concentration. Hardness was determined at each renewal period in fresh and old media, in the control and in the highest test item concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily
- Light intensity: within the range of 15-20 µE m^2/s

EFFECT PARAMETERS MEASURED: EC50, NOEC and LOEC for reproduction, mortality and growth

TEST CONCENTRATIONS
- Test concentrations for range finding study: 0, 6.25, 12.5, 25, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

37.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95%-CI: 26.8-52.9

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- No mortality was observed in any of the test groups
- There was a statistically significant difference between the reproductive output of the parent animals per day at the two highest concentration groups of 20 and 40 mg/L compared to the control (Dunnett's Test, α=0.05).
- Aborted eggs, dead offspring, males or ephippia were not found during the study.
- The average cumulative number of young per parent animal in the control was 199.2 with a variation coefficient of 24.05%
- Reproductive output of parent animals and reproductive output per animal were both significantly reduced at the highest test concentrations (20 and 40 mg/L).

Reported statistics and error estimates:

For the assessment of the reproductive output of the parent animals analysis of variance (AN OVA) was performed and the mean for each concentration was compared with the control mean using an appropriate multiple comparison method (Dunnett's test). For the analysis of the mean body length of test animals the Dunnett's tests was used by TOXSTAT (version 3.5) software.
Mortality was not observed neither in the control and in any test item treated groups during the study, therefore statistical analysis was not performed on the mortality data.

Table 1 Measured concentrations

	Nominal concentration (mg/L)	Measured concentration (mg/L) with 95% confidence interval	Percentage of nominal
27 April 2012	Control	n.d.	n.a.
	2.5	2.51± 0.03	100
	5.0	5.02± 0.02	100
	10	10.3± 0.44	103
	20	20.4± 0.06	102
	40	40.3± 0.15	101
30 April 2012	Control	n.d.	n.a.
	2.5	2.92± 0.35	117
	5.0	4.90± 0.12	98
	10	9.63± 0.12	96
	20	19.7± 0.73	99
	40	38.5± 1.09	96
09 May 2012	Control	n.d.	n.a.
	2.5	2.51± 0.01	100
	5.0	5.04± 0.02	101
	10	10.1± 0.02	101
	20	20.0± 0.03	100
	40	39.5± 0.15	99
11 May 2012	Control	n.d.	n.a.
	2.5	2.58± 0.18	103
	5.0	4.99± 0.11	100
	10	9.94± 0.21	99
	20	20.4± 0.79	102
	40	10.0± 0.81	100
16 May 2012	Control	n.d.	n.a.
	2.5	2.45± 0.04	98
	5.0	4.99± 0.07	100
	10	10.1± 0.11	101
	20	20.4± 0.09	102
	40	40.7± 0.21	102
18 May 2012	Control	n.d.	n.a.
	2.5	2.43± 0.13	97
	5.0	4.72± 0.06	94
	10	9.38± 0.16	94
	20	19.2± 0.75	96
	40	38.0± 0.55	95

n.d.: not detected

n.a.: not applicable

Table 2 Summary of reproductive output of the parent animals

Concentration (mg/L)	Mean number of living offspring/parent animal	SD	CV (%)	Reduction (%)	Significance
Control	119.2	24.1	20.18	-	-
2.5	108.8	40.6	37.27	8.7	n.s.
5	97.9	35.9	36.29	17.9	n.s.
10	94.1	14.7	15.58	21.1	n.s.
20	75.4	41.8	55.42	36.7	*
40	56.6	21.6	38.15	52.5	*

SD         : standard deviation
CV (%) : variation coefficient
*            : statistically significantly different compared to the control (Dunnett's Test,α=0.05)
n.s.:     : statistically not significantly different compared to the control (Dunnett's Test,α=0.05)

Table 3 Reproductive output of the parent animals per day

Concentration (mg/L)	Mean number of living offspring/parent animal	SD	CV (%)	Reduction (%)	Significance
Control	8.45	1.7	19.66	-	-
2.5	8.11	3.1	37.87	4.0	n.s.
5	6.99	2.6	36.74	17.2	n.s.
10	6.67	1.0	15.25	21.0	n.s.
20	5.45	3.0	55.83	35.5	*
40	4.14	1.6	37.58	51.0	*

SD         : standard deviation
CV (%) : variation coefficient
*            : statistically significantly different compared to the control (Dunnett's Test,α=0.05)
n.s.:     : statistically not significantly different compared to the control (Dunnett's Test,α=0.05)

Validity criteria fulfilled:

yes

Remarks:

There was no mottality in the control group. The mean number of live offspring per parent animal surviving in the control at the end of the test was 119.20 with a variation coefficient of24.05 %. No ephippia was observed during the test.

Conclusions:

Under the conditions of the present study Equamide M100 did not cause mortaility or affect growth of Daphnia magna. The 21-day LC50 of parents was > 40 mg/L. Reproduction was affected at 20 and 40 mg/L, the 21-day EC50 for reproduction was 37.6 mg/L. The overall NOEC was 10 mg/L.

Executive summary:

A study was performed to assess the effect of Equamide M100 on the reproduction of Daphnia magna.The method followed that described in the OECD TG No 211.

A stock solution was prepared at 40 mg/L and stirred mechanically, this solution was used ad the highest concentration. Lower concentrations used in the final test were prepared by dilution and were 2.5, 5.0, 10.0 and 20.0 mg/L. 10 daphnids (< 24 hours) per concentration were exposed for 21 days, under semi-static conditions. Test solutions were renewed every 2 -3 days, based on the stability of test item in test medium. Samples were taken from fresh solutions on day 0, 12 and 19 and from old solutions on day 3, 14 and 21 and were analysed to determine actual concentrations of test item. Results showed measured concentrations were 94 -117% of nominal concentrations. Consequently, the results were based on nominal concentrations.

No mortality was observed in the control and any of the treatment groups. Reproduction was statistically significant reduced at 20 and 40 mg/L. The 21 -day EC50 for reproduction was 37.6 mg/L. No effect on growth was observed. Under the conditions of the present study Equamide M100 did not affect reproduction and growth of Daphnia magna at 10 mg/L after 21 days of exposure (NOEC).

The study met the acceptability criteria and was considered valid.

Description of key information

A study was performed to assess the effect of Equamide M100 on the reproduction of Daphnia magna. The method followed that described in the OECD TG No 211.

A stock solution was prepared at 40 mg/L and stirred mechanically, this solution was used ad the highest concentration. Lower concentrations used in the final test were prepared by dilution and were 2.5, 5.0, 10.0 and 20.0 mg/L. 10 daphnids (< 24 hours) per concentration were exposed for 21 days, under semi-static conditions. Test solutions were renewed every 2 -3 days, based on the stability of test item in test medium. Samples were taken from fresh solutions on day 0, 12 and 19 and from old solutions on day 3, 14 and 21 and were analysed to determine actual concentrations of test item. Results showed measured concentrations were 94 -117% of nominal concentrations. Consequently, the results were based on nominal concentrations.

No mortality was observed in the control and any of the treatment groups. Reproduction was statistically significant reduced at 20 and 40 mg/L. The 21 -day EC50 for reproduction was 37.6 mg/L. No effect on growth was observed. Under the conditions of the present study Equamide M100 did not affect reproduction and growth of Daphnia magna at 10 mg/L after 21 days of exposure (NOEC).

The study met the acceptability criteria and was considered valid.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10 mg/L