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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:
From 2000-08-08 to 2000-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
All validity criteria were fulfilled.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
- Water solubility: 13.7 g/L at 20 °C [Experimental data (Safepharm, 2008), OECD 105 (flask method)]
- Vapour pressure: 0.178 Pa at 25 °C [Experimental data (Harlan, 2009), analogue data, OECD 104 (gas saturation method)]
- Log Kow: 1.95 at 30 °C [Experimental data (Safepharm, 2008), OECD 117 (HPLC method)]
- Relative density: 1.35 at 20°C [Experimental data (Safepharm, 2008), OECD 109 (pycnometer method)]
- Stability in water: Hydrolytically stable at environmentally relevant pHs (internal experimental Sponsor data)
Analytical monitoring:
yes
Details on sampling:
Concentrations of test item in the exposure system were determined at 0 and 48 hours of the toxicity test from samples collected from the exposure system. At test initiation a 10.0-mL sample was collected from each parent test solution. At test termination, the replicate test solutions were composited and a 10.0-mL sample was collected from each treatment composite. Control and test item fortified QC samples were also analyzed at each sample point.
Quality control samples were prepared by fortifying water collected from the control chamber with test item at concentrations of 6.0 and 110 mg/L. The analysis of the exposure system samples for test item during the Daphnia toxicity test was accomplished following the procedures and conditions outlined for the analysis of the stability and method validation studies.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary standard was prepared by diluting 0.2011 g of test item with dilution water to a volume of 2000 mL. The primary standard was sonicated for approximately 20 minutes and then magnetically stirred for approximately 20 minutes to dissolve the test substance. Volumes of 62.5, 125, 250, and 500 mL from the primary standard were diluted with dilution water to provide total volumes of 1000 mL for preparation of treatment levels - 6.25, 12.5, 25.0 and to 50.0 mg/L.
- Eluate: Dilution water
- Controls: One control. Dilution water without test item.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: ABC Laboratories' in-house culture
- Age at study initiation: < 24 h
- Method of breeding: The adult daphnids that produced the neonates were cultured in a temperature-controlled water bath at approximately 20 °C.

ACCLIMATION
- Acclimation period: The adult daphnids were isolated from the culture and transferred to dilution water < 24 hours prior to the beginning of the definitive test.
- Feeding: During the holding period, the adult daphnids received a diet of algae (Selenastrum capricornutum and/or Ankistrodesmus falcatus) supplemented by a prepared artificial invertebrate food.
- The adult daphnids used in the definitive test were cultured for approximately 16 days and were considered acceptable with no signs of stress, disease, or physical damage.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
148 mg/L as CaCO3.
Test temperature:
20 ± 1°C
pH:
8.46-8.64
Dissolved oxygen:
7.81-8.47 mg O2/L (92-97 % saturation)
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentration: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L
- Measured concentration: 5.58, 11.6, 23.4, 47.1 and 96.2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Eight-ounce glass jars which were covered with plastic petri dish covers, Each test chamber measured approximately 7.0 cm in diameter by 8.5 cm in height. Solution depth was approximately 6.2 cm, for a volume of approximately 200 mL.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a laboratory blended freshwater that originated from a deep well. A portion of this well water was passed through a reverse osmosis system and then blended back with raw well water to achieve hardness between 130 to 160 mg/L as CaCO3. The dilution water was biologically aged and passed through a cartridge filter and ultraviolet sterilizer before use in the test.
- Alkalinity: 160 mg CaCO3/L
- Conductivity: 316 µs

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Fluorescent lighting was maintained on a 16-hour day-light photoperiod with 30-minute simulated dawn and dusk periods.
- Light intensity: 632.1 to 698.6 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations of immobilisation and/or mortality were recorded at 24 and 48 hours.

TEST CONCENTRATIONS
- Range finding study: A preliminary range-finding test was conducted under static conditions at 0.10, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: After 48 hours of exposure, the percent immobilization was 0% at concentrations ≥ 0.10 mg/L. Immobilization was 60% in the control. The observed immobility in the control was considered an aberrant effect. Based upon these results, the definitive test was conducted at nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100 mg/L.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 96.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: As reported in the study report
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Measured conc. >80% of nominal
Duration:
48 h
Dose descriptor:
other: Highest tested concentration without observed effect
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: no sub-lethal effects
Details on results:
- Behavioural abnormalities: None
- Mortality of control: None
- After 48 hours of exposure immobilization of Daphnia magna for the definitive test was 0% in the control and all test item treatments. Quiescence was observed at the 23.4 mg/L treatment where two of the 20 exposed daphnids were quiescent.

See results of the definitive test in the Table 6.1.3/1 in "Any other information on results incl. tables".
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None

The measured concentrations of test item at 0 hour ranged from 5.47 to 99.6 mg/L. The measured concentrations of test item at 48 hours ranged from 5.69 to 92.7 mg/L. The overall mean measured concentrations ranged from 5.58 to 96.2 mg/L and were between 89 and 96% of the nominal test concentrations. Controls measured at 0 and 48 hours were reported as less than the minimal quantifiable limit (MQL) or <0.500 and <1.25 mg/L, respectively. All test solutions were clear and colorless with no visible particulate material surface film, or undissolved test material for the duration of the definitive test.

Two quality control (QC) spikes were prepared and measured at each sampling period. The measured QC spike concentrations at 0 hour were 4.80 and 103 mg/L which were 80 and 94% of nominal concentrations respectively. The measured QC spike concentrations at 48 hours were 5.14 and 104 mg/L which were 86 and 95% of nominal concentrations, respectively.

Table 6.1.3/1: Immobilization of Daphnia magna Exposed to test item for 48 Hours Under Static Test Conditions

Nominal concentrations (mg/L)

Replicate

Cumulative Number immobile (% Immobilization)

24 hours

48 hours

Total/Treatment

Control

A

0 (0)

0 (0)

0 (0)

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

6.25

A

0 (0)

0 (0)

0 (0)

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

12.5

A

0 (0)

0 (0)

0 (0)

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

25.0

A

0 (0)

0* (0)

0 (0)

B

0 (0)

0* (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

50.0

A

0 (0)

0 (0)

0 (0)

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

100

A

0 (0)

0 (0)

0 (0)

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

 

*One of the surviving daphnids was quiescent.

Note: Unless otherwise noted, all daphnids were normal in appearance.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the 48 h-EC50 for test item to Daphnia magna is > 100 mg/L, based on analytically confirmed nominal concentrations.
Executive summary:

This study was performed according to OECD Guideline 202 with GLP statement, to assess the 48h-acute toxicity of the of the test substance to Daphnia magna, under static conditions.

In this study, test item was exposed to daphnids at the concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg/L for 48 hours. The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The dilution water was freshwater possessing a total hardness of 148 mg/L CaCO3. The test chambers were randomly positioned in a temperature-controlled water bath that maintained the temperature of the test solutions between 19 and 22 °C during the test. Before definitive test, a 48-hour static range-finding test was conducted at the nominal concentrations of 0 (control), 0.10, 1.0, 10, and 100 mg/L and the percent immobilization was 0% at concentrations ≥0.10 mg/L.

 

The mean measured concentrations of test item were 5.58, 11.6, 23.4, 47.1, and 96.2 mg/L, i.e., 89 to 96% of the targeted nominal concentrations. The test substance was considered to be soluble and stable under test conditions based upon the results of the stability study and the definitive measured concentrations.

 

After 48 hours of exposure immobilization of Daphnia magna for the definitive test was 0% in the control and all test item treatments. Quiescence was observed at the 23.4 mg/L treatment where two of the 20 exposed daphnids were quiescent. The calculated 48-hour EC50 value was was mentioned in the study report as > 96.2 mg/L based on measured concentrations. However, as the concentrations of the test substance has been satisfactorily maintained within +/-20% of the nominal concentrations throughout the test, the result can be based on nominal concentrations, so >100 mg/L.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) > 100 mg/L (analytically confirmed nominal concentration).

Key value for chemical safety assessment

Additional information

One key study is available to assess the 48h acute toxicity of the registered substance to Daphnia magna, under static conditions, according to the OECD Guideline 202 with GLP statement. Following a preliminary range-finding test, twenty daphnids (four replicates per treatment, five daphnids per replicate) were exposed to the test substance at the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg/L for 48 hours, in a temperature-controlled water bath that maintained the temperature of the test solutions between 19 and 22°C. The no-treatment control daphnids were exposed to dilution water only. The mean measured concentrations of test substance were 5.58, 11.6, 23.4, 47.1, and 96.2 mg/L, i.e., 89 to 96% of the targeted nominal concentrations. The test substance was considered to be soluble and stable under test conditions based upon the results of the stability study and the definitive measured concentrations. After 48 hours of exposure, immobilization of Daphnia magna for the definitive test was 0% in the control and all test item treatments. Quiescence was observed at the 23.4 mg/L treatment where two of the 20 exposed daphnids were quiescent. The calculated 48-hour EC50 value was mentioned in the study report as > 96.2 mg/L based on measured concentrations. However, as the concentrations of the test substance has been satisfactorily maintained within +/-20% of the nominal concentrations throughout the test, the result can be based on nominal concentrations, so > 100 mg/L.