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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental dates: Initiation 17 October 2013; Termination 07 November 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study conducted according to Good Laboratory Practices. Measured concentrations were substantially lower than nominal concentrations. The maximum acceptable toxicant concentration could not be defined due to low water solubility and rapid disappearance of the test substance.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
Daphnids ere offered algae once on day 0 and twice on study days 17 and 18. Daphnids were not fed the day of test termination. There were no significant deviations.
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
yes
Remarks:
Daphnids ere offered algae once on day 0 and twice on study days 17 and 18. Daphnids were not fed the day of test termination. There were no significant deviations.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The concentration of test material was measured in test solution samples collected on days 0, 7, 14, and 21. At each sampling time point, samples were collected and prepared from each of the four replicate test chambers for the negative control, vehicle control, and each treatment level. Since the test substance may be adversely affected by the test organisms or the diet provided in the test chambers, additional samples were collected from the splitter cups and the chemical mixing box at each sampling time point. Also a sample was collected from the stock solution.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Diluter stock solution was prepared using dimethylformamide (DMF) as a solvent for the test substance.
- Controls: yes (water control and vehicle control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): The concentration of solvent in the prepared test solution was 25.5 µL DMF/L. The vehicle control and all test substance treatment solutions contained an approximately equal concentration of DMF.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In-house culture
- Age of parental stock (mean and range, SD): <24 hours old
- Feeding during test: During the exposure daphnids were offered an algal suspension (3.0 x 10E7 cells/mL Pseudokirchneriella subcapitata) and a prepared invertebrate food solution (YCT daphnid feed mixture) containing approximately 2.5 mg of solids/mL. At least 4 mL of the algal suspension were manually added to each replicate chamber four times daily (e.g., early morning, late morning, early afternoon, and late afternoon), except on the following occasions: 1) the day of test initiation (i.e., day 0) when 8 mL was added to each replicate on just one occasion, 2) days 13, 15, 16, 19, and 20 when 6 mL was offered as the last of the four feedings at the end of the work day, and 3) on days 17 and 18 when just two feedings of 4 and 6 mL were offered. The invertebrate food solution was manually added to each replicate chamber four times daily, except on the day of test initiation when 2 mL was added to each replicate on just one occasion. The volume of the prepared invertebrate food solution offered at each feeding was 1 mL. The daphnids were not fed the day of test termination (i.e., day 21).


ACCLIMATION
- Acclimation period: No, since the culturing and testing environmental parameters were equivalent (i.e., temperature, dilution water, and lighting)

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
156 CaC03/L
Test temperature:
20 ± 1°C
pH:
8.1 to 8.4
Dissolved oxygen:
7.2 to 8.6 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0.065, 0.13, 0.25, 0.50 and 1.0 mg a.i./L
Measured concentraitons: 0.00638, 0.0162, 0.0266, 0.0428, and 0.121 mg a.i./L

Mean measured concentrations for the test substance treatments were 0.00638, 0.0162, 0.0266, 0.0428, and 0.121 mg a.i./L, which represented 9 to 12% of the nominal concentrations. Mean measured concentrations for the test substance in the splitter cups were 21 to 44% of the nominal concentrations. Measured concentrations in samples from the chemical mixing box ranged from 31 to 56% of the nominal concentration and measured concentrations from stock solution samples ranged from 84 to 93% of nominal. See "Overall Remarks" for more details.

Biological resullts were based on the mean measured concentrations from the replicate solutions since measured concentrations were not within 20% of the nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Test chambers consisted of 1-L glass beakers with a screened overflow notch. Each test chamber measured approximately 15 cm high and 10 cm in diameter with test solution depth maintained at approximately 12 cm.
- Type of flow-through (e.g. peristaltic or proportional diluter): Intermittent-flow proportional diluter system. The diluter system was designed in a manner to achieve a concentration of solvent in each test substance treatment that was equal to the concentration of solvent in the vehicle control.
- Renewal rate of test solution (frequency/flow rate): Six volume additions to the test chamber per 24 hour period.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a moderately hard freshwater prepared by blending naturally hard well
water with well water that was demineralized by reverse osmosis (RO). The well water and RO water was blended to yield a total hardness of 130 to 160 mg CaC03/L. Prior to delivery to the test chambers, the dilution water was aerated and passed through a sediment filter and UV sterilizer.
- Total organic carbon: <2.0 mg/L
- Alkalinity: 152 to 154 CaC03/L
- Conductivity: 346 to 348 µS
- Culture medium different from test medium: No


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light:8-hour dark photoperiod with a 30-minute simulated dawn and dusk transition period
- Light intensity: 458 to 552 lux (measured on Day 21)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations were made daily on the number of surviving adult daphnids, occurrence of abnormalities, and production of neonates. Immobile daphnids, defined as those organisms not able to swim within 15 seconds after gentle agitation of the test vessel or gentle disturbance of the individual, were discarded; therefore, immobility was synonymous with mortality. At test termination, the length (head to base of spine) of each surviving adult was measured by use of a calibrated ocular micrometer of a Leica S6E microscope. After length measurements, the daphnids from each replicate were pooled and placed in pre-weighed aluminum pans and dried for approximately two days at approximately 60°C. After drying, the aluminum pans were placed in a desiccator to cool and then weighed.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: A range-finding test was not conducted. The highest target nominal test concentration definitively tested was to be approximately equal to the 48-hour EC50 for 9-DAME to Daphnia magna (i.e., 0.80 mg/L; as provided by Sponsor)
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: mortality, reproduction, growth
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.121 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: mortality, reproduction, growth
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.121 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: mortality, reproduction, growth
Remarks on result:
other: The maximum acceptable toxicant concentration could not be defined due to low water solubility and rapid disappearance of the test substance
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.121 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
- Mortality of parent animals: There was no statistically significant decrease of survival, as compared to the pooled control (untreated and vehicle groups), in any of the test substance treatments.
- No. of offspring produced per day per female: The number of young per reproductive day was 9.4 and 9.0 in the control and vehicle control, respectively, and ranged from 8.0 in the 0.0266 mg a.i./L treatment to 9.0 in the 0.0428 mg a.i./L treatment. The pooled control young per reproductive day was 9.2. There was a significant reduction in young per surviving adult and young per adult reproductive day in the 0.0266 mg a.i./L test substance treatment, but these reductions were not considered to be concentration-dependent, based on a lack of significant reduction at all higher test treatment levels.
- Body length and weight of parent animals: No statistically significant reduction in mean dry weight as compared to the pooled control was detected for all test substance treatments with the exception of the 0.0266 mg a.i./L treatment. The statistically significant weight reduction in the 0.0266 mg a.i./L treatment was not considered to be biologically significant since there were no significant effects at all higher concentrations. There was no statistically significant reduction in length in any of the test substance treatments as compared to the pooled control.
- Type and number of morphological / behavioural abnormalities: One daphnid was observed trailing extraneous matter in the 0.0428 mg a.i./L test substance treatment on study day 17 and throughout the remainder of the test. No other sublethal effects were observed.
- Number of males and females (parental):
- Time to first brood release or time to hatch: The average number of days until first brood was eight in the negative control, vehicle control, and all test substance treatments.

Data tables are shown below for survival and for endpoints that had significant intergroup differences.

Survival Observed During a 21-Day Chronic Flow-Through Toxicity Test with Daphnia magna Exposed to 9-Decenoic Acid Methyl Ester

Mean Measured Concentration
(mg a.i./L)

REP

Start Number

Day 21 Number Alive

Treatment % Survival

0 (Control)

A

10

9

93

B

10

10

C

10

10

D

10

8

0 (Vehicle Control)

A

10

10

85

B

10

9

C

10

7

D

10

8

Pooled Control

 

--

--

89

0.00638

A

10

10

98

B

10

9

C

10

10

D

10

10

0.0162

A

10

10

85

B

10

10

C

10

8

D

10

6

0.0266

A

10

10

98

B

10

9

C

10

10

D

10

10

0.0428

A

10

10

100

B

10

10

C

10

10

D

10

10

0.121

A

10

10

93

B

10

10

C

10

9

D

10

8

Notes:Ten daphnids were placed in each replicate at initiation. Percent values were rounded to the nearest whole number. There was no statistically significant reduction in survival as compared to the pooled control.

Number of Young Produced During a 21-Day Chronic Flow‑Through Toxicity Test with Daphnia magna Exposed to 9-Decenoic Acid Methyl Ester

Mean Measured Concentration
(mg a.i./L)

REP

Number of Parents at

Study Start

Young/Parent
at Study Start

Number of Parents Surviving at

Day 21

Young per Surviving Parent

Young per Adult Reproductive Day

Control

A

10

119

9

132

8.8

B

10

145

10

145

9.7

C

10

133

10

133

8.9

D

10

136

8

169

10.0

Total

40

133

37

145

9.4

Vehicle Control

A

10

129

10

129

8.6

B

10

118

9

131

8.6

C

10

103

7

147

9.8

D

10

119

8

149

8.9

Total

40

117

34

139

9.0

Pooled Control

--

125

--

142

9.2

0.00638

A

10

127

10

127

8.5

B

10

122

9

135

8.3

C

10

128

10

128

8.6

D

10

131

10

131

8.7

Total

40

127

39

130

8.5

0.0162

A

10

143

10

143

9.5

B

10

141

10

141

9.4

C

10

107

8

134

8.6

D

10

84.7

6

141

8.1

Total

40

119

34

140

8.9

0.0266

A

10

101

10

101

6.7

B

10

125

9

138

8.4

C

10

132

10

132

8.8

D

10

123

10

123

8.2

Total

40

120

39

124*

8.0*

0.0428

A

10

133

10

133

8.9

B

10

119

10

119

7.9

C

10

148

10

148

9.9

D

10

143

10

143

9.5

Total

40

136

40

136

9.0

0.121

A

10

145

10

145

9.7

B

10

117

10

117

7.8

C

10

124

9

138

8.6

D

10

108

8

136

8.8

Total

40

124

37

134

8.7

* Statistically significant reduction as compared to the pooled control.

 Note:  Although there was a significant reduction in young per surviving adult and young per adult reproductive day in the 0.0266 mg a.i./L test substance treatment, these reductions were not considered to be concentration-dependent, based on a lack of significant reduction at all higher test treatment levels.

Mean Dry Weight of First Generation Daphnia magna After a 21‑Day Chronic Flow-Through Toxicity Test with Daphnia magna Exposed to 9‑Decenoic Acid Methyl Ester

Mean Measured

Conc.

(mg a.i./L)

 

Mean Dry Weight Per Organism in mg (N)a

Replicate A

 

Replicate B

 

Replicate C

 

Replicate D

 

Treatment Meanb

Control

 

0.61 (9)

 

0.70 (10)

 

0.60 (10)

 

0.83 (8)

 

0.68

Vehicle Control

 

0.70 (10)

 

0.64 (9)

 

0.71 (7)

 

0.70 (8)

 

0.69

Pooled Control

 

---

 

---

 

---

 

---

 

0.69

0.00638

 

0.67 (10)

 

0.59 (9)

 

0.59 (10)

 

0.70 (10)

 

0.64

0.0162

 

0.80 (10)

 

0.63 (10)

 

0.59 (8)

 

0.62 (6)

 

0.66

0.0266

 

0.50 (10)

 

0.68 (9)

 

0.52 (10)

 

0.48 (10)

 

0.54*

0.0428

 

0.68 (10)

 

0.63 (10)

 

0.58 (10)

 

0.60 (10)

 

0.62

0.121

 

0.70 (10)

 

0.61 (10)

 

0.66 (9)

 

0.74 (8)

 

0.68

a    N = number of organisms weighed.

b    The weighted mean is calculated by dividing the sum of all replicates within treatment by the total number of organisms weighed for the treatment.

 

* Statistically significant reduction as compared to the pooled control.

 

Note:  Although there was a significant reduction in mean dry weight per organism in the 0.0266 mg a.i./L test substance treatment, this reduction was not considered to be concentration-dependent based on a lack of significant reduction at all higher test treatment levels.

Validity criteria fulfilled:
yes
Remarks:
The relevant test acceptability criteria were met for this study.
Conclusions:
No significant concentration-dependent adverse biological effects were observed for any concentration for any endpoint. Based on nominal concentrations, the 21-day NOEC and LOEC for Daphnia magna exposed to the test substance was 0.121 and >0.121 mg a.i./L. respectively, based on all parameters. The MATC could not be calculated. The 21-day EC50 for adult survival was estimated to be >0.121 mg a.i./L, the highest treatment tested.
Executive summary:

The chronic toxicity of 9-decenoic acid methyl ester to Daphnia magna was evaluated under flow-through conditions in a guideline study (OECD 211; OPPTS 850.1300) conducted in accordance with Good Laboratory Practices. Diluter stock solutions were prepared using dimethylformamide as a solvent for the test substance. The mean measured concentrations of test substance were substantially lower than nominal concentrations. In the test chambers, measured concentrations were 5 to 21 % of nominal, even though solutions were being changed at about six volumes per day. No significant concentration-dependent adverse biological effects were observed for any concentration for any endpoint. Based on measured concentrations, the 21-day NOEC and LOEC were 0.121 and >0.121 mg a.i./L, respectively. Expressed as nominal or loading rates, the NOEC can be estimated as 1.0 mg a.i./L. The maximum acceptable toxicant concentration could not be defined due to low water solubility and rapid disappearance of the test substance.

The large gap between nominal and mean measured concentrations may make interpretation of the test results problematic.

Description of key information

Key study performed according to GLP and Guidelines (OECD 211, OPPTS 850.1300)

The chronic toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated.

Mean measured concentrations of test substance were substantially lower than nominal concentrations.

21-day NOEC (measured) = 0.121 mg/L

21-day NOEC (nominal) = 1.0 mg/L

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.121 mg/L

Additional information

The chronic toxicity of 9-decenoic acid methyl ester to Daphnia magna was evaluated under flow-through conditions in a guideline study (OECD 211; OPPTS 850.1300) conducted in accordance with Good Laboratory Practices. 

No significant concentration-dependent adverse biological effects were observed for any concentration for any endpoint: adult mortality, reproduction, or growth (length and weight).

 

Based on nominal concentrations, the 21-day NOEC was 1.0 mg a.i./L. However, measured concentrations were not within 20% of the nominal concentrations, so mean measured concentrations in the replicate solutions are reported. Based on mean measured concentrations, the 21-day NOEC and LOEC were 0.121 and >0.121 mg a.i./L, respectively based on all parameters. No meaningful 21-day EC50 can be calculated because no adverse effects were observed.

 

The flow-through conditions did not resolve the problem with rapid disappearance of test material as seen in previous short-term tests. Diluter stock solutions were prepared using dimethylformamide as a solvent. Concentrations in the diluter stock solution was close to expected. Flow rates were maximized (six volume additions to each test chamber in a 24-hour period). Functioning of the diluter system was confirmed with saline solution. Flow-through operation was initiated 4 days before test initiation to pre-treat test chambers.

 

The mean measured concentrations in test chambers were only 5 to 21% of nominal. Nominal and (measured) concentrations (in mg a.i./L) were: 0.065 (0.00638), 0.13 (0.0162), 0.25 (0.0266), 0.5 (0.0428) and 1.0 (0.121). Loss of test substance was observed even in the initial mixing chamber, with concentrations being 31 to 56% of nominal. The large gap between nominal and measured concentrations makes interpretation of the test results problematic.

 

The maximum acceptable toxicant concentration could not be defined due to low water solubility and rapid disappearance of the test substance.

 

Based on the apparent instability of the test material and this decline/disappearance during the test, it is considered that the test should not be regarded reliable without restriction. Due to this, the results from this study have not been used to further refine PNEC’s or to further review the environmental classification of the substance.

 

Efforts to achieve stable test substance concentrations have been performed. Factors such as volatilization, photodegradation, hydrolysis and biodegradation do not appear to control the inherent property of the test substance to leave the water column. Sorption to surfaces, silicone adhesive and organic material do appear to have a role. The results of any repeated testing that achieves stability of the test material in water will be compared to this study and the existing short-term study to refine PNECs and environmental classification as appropriate.

An acute Daphnia magna study using a passive dosing technique (O-rings) has now been conducted and obtained stable concentrations throughout the test duration. This acute study is therefore considered more relevant for environmental assessment than the Daphnia magna reproduction study.