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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-01 to 2012-03-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were collected from one test chamber of the 37 and 73 ng/L treatment groups one day prior to the start of the test after conditioning the diluter for approximately two days. Samples also were collected from alternating replicate test chambers of the 37 and 13 ng/L treatment groups and the negative control group at the beginning of the test and at 48 hours (± I hour) to measure concentrations of the test substance. The test solution samples were collected, placed in glass volumetric flasks, and processed immediately for analysis. Samples of all stock solutions were also collected for analysis prior to test initiation and at the end of the test, to confirm delivery of the test substance to the test system.
Vehicle:
yes
Details on test solutions:
Individual stock solutions were prepared for each of the five concentrations tested. All test solutions were prepared as is, with no adjustment for active ingredient during preparation.

A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimelhylformamide (DMF) at a nominal concentration of 3.65 μg/mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 0.23, 0.46, 0.90 and 1.85 μg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion and were clear and colorless. Aliquots of each stock sufficient to last for Ihe entire test period were placed in the syringe pump at the initiation of the test solution flows; the volume of stock solution consumed was measured every two to three days during the study. Remaining stock solutions were stored ambient in glass volumetric flasks.

The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 3.1 μL/minute where they were mixed with dilution water delivered at a rate of 155 mL/minute to achieve the desired test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all treatment groups was 0.02 mL/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnid neonates used in the test were less than 24 hours old at test initiation and were obtained from cultures maintained by the testing laboratory.

Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.8 to 20.9°C, the pH of the water ranged from 8.3 to 8.4 and dissolved oxygen concentrations were ≥7.8 mg/L (≥87% of saturation).

Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. The adults were fed prior to test initiation, but neonates were not fed during the test.

The eleven adult daphnids used to supply neonates for the test were held for at least 23 days prior to collection of the juveniles for testing and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 1-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 10 daphnids. Each group of daphnids then was transferred to the test compartment in an indiscriminately assigned test chamber to initiate the test. All transfers were made below the water surface using wide-bore pipettes.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
148 mg/L as CaCO3
Test temperature:
20+/-1°C
pH:
8.0 to 8.2.
Dissolved oxygen:
≥8.3 mg/L (≥92% of saturation)
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control), 4.6, 9.1, 18, 37 and 73 ng/L

Mean measured concentrations in the two highest treatments (37 and 73 ng/l): 3.5 and 19 ng/L (9.5 and 26% of nominal)
Details on test conditions:
Exposure system: The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance, a solvent control and a negative control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, Massachusetts) were used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately five volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test chambers.

The syringe pumps used to deliver stock solutions or solvent to the mixing chambers and the rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test. The proportion of the test water that was split into each replicate test chamber was checked prior to the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the two replicates. Delivery of test solutions to the test chambers was initiated three days prior to test initiation in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.

The test chambers were placed in a temperature-controlled water bath to maintain the target water temperature throughout the test period. Test chambers were 25-L Teflon-lined stainless steel aquaria filled with approximately 22 L of test water. The depth of the test water in a representative chamber was 28.3 cm. Each test chamber contained one test compartment constructed from a glass beaker approximately 6.5 cm in diameter and 12 cm in height, with nylon screen attached to two holes on the sides of the beaker. The depth of the test water in a representative compartment was 10.2 cm. All test chambers were labelled with the project number, test concentration and replicate designation.

Environmental Conditions: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test.

The target test temperature during the study was 20 ± 1°C. Temperature was measured in each test chamber at the beginning and end of the test. Temperature also was monitored continuously in one negative control test chamber.

Dissolved oxygen and pH were measured in one replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24 hours, and at the end of the test, with measurements typically alternating between replicates in each group at each measurement interval.

Hardness, alkalinity, specific conductance and total organic carbon (TOC) in the dilution water at the beginning of the test were measured.

Observations: All organisms were observed periodically to determine the number of immobile organisms in each treatment group. Immobility was defined as a lack of movement by the organism except for minor activity of the appendages. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24 and 48 hours after test initiation.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 19 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: 0
- Mortality of control: 0
- Other adverse effects control: 0
Reported statistics and error estimates:
The absence of immobile daphnids in any of the test substance treatment groups during the test precluded the statistical calculation of EC50 values at 24 and 48 hours. Therefore, the EC50 values were estimated to be greater than the highest concentration tested. The no-immobility concentration and the no-observed-effect concentration (NOEC) were determined by visual interpretation of the immobility and observation data.

Table 1. Test results

Nominal concentration (ng/l)

Mean measured concentration (ng/l)

Measured concentration as % of nominal

Immobilisation after 4 hours (%)

Immobilisation after 24 hours (%)

Immobilisation after 48 hours (%)

0 (Control)

<LOQ

-

0

0

0

0 (Solvent control)

<LOQ

-

0

0

0

4.6

Not determined

-

0

0

0

9.1

Not determined

-

0

0

0

18

Not determined

-

0

0

0

37

3.5

9.5

0

0

0

73

19

26

0

0

0

Validity criteria fulfilled:
yes
Conclusions:
A 48-hour EC50 value of >19 ng/l and NOEC of ≥19 ng/l have been determined for the effects of the test substance on mobility of Daphnia magna. The test substance has a water solubility of approximately 7.3 ng/l and therefore, the results indicate that it is not toxic at its solubility limit.

Description of key information

EC50 (48-h): >19 ng/l mobility of Daphnia magna, reliability 1 (Wildlife International Ltd., 2012)

Key value for chemical safety assessment

Additional information

A 48 hour EC50 value of >19 ng/l (highest concentration tested) has been determined for the effects of the test substance on mobility of Daphnia magna in accordance with OECD TG 202, and in compliance with GLP (Wildlife International, 2012). Due to the slow hydrolysis of the test substance and the test media preparation and exposure method, it is likely that the test organisms were predominantly exposed to the parent substance. The test substance has a water solubility of <10 ng/l and therefore the results indicate that it is not toxic at its solubility limit.