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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Prior to test initiation, a 1000 μg/mL primary stock solution was prepared by placing 0.0256 g of test substance in a 25-mL volumetric flask and bringing it to volume with dimethylformamide (DMF, CAS No. 68-12-2). The resultant stock solution was observed to be clear and colorless with no visible undissolved test substance following preparation. Secondary stock solutions were prepared from dilutions of the primary stock and were observed to be clear and colorless with no visible undissolved test substance. Exposure solutions were prepared at test initiation and daily thereafter.
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 ml/L
- Evidence of undissolved material: all test solutions were observed to be clear and colorless with no visible undissolved test substance

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: laboratory cultures maintained at Springborn Smithers
- Age of parental stock: < 24 h
- Feeding during test:
The food was introduced at a rate of 200 μL of algal suspension (Ankistrodesmus falcatus, 4 x 107 cells/mL), and 50 μL of a yeast, cereal leaves and digested flaked fish food (YCT) suspension, once daily. This quantity of algal suspension (200 μL/daphnid/day) is equivalent to approximately 0.21 mg carbon/daphnid/day.
ACCLIMATION
- Acclimation conditions: same as test
- Type and amount of food: During culture, daphnids were fed various amounts based on their age. Daphnids that were 0 to 6 days old were fed 1.0 mL of algae (Ankistrodesmus falcatus, 4 x 107 cells/mL) and 0.5 mL of YCT (yeast, cereal leaves and flaked fish food) suspension per vessel per day. Daphnids that were 7 to 10 days old were fed 1.5 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were > 10 days old were fed 2.0 mL of algae and 0.5 mL of YCT suspension per vessel per day. Representative samples of the food source were periodically analyzed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed. Based on these analyses, the food source was considered to be of acceptable quality since all analyte concentrations were below levels of concern.
- Health during acclimation: The adult daphnids used to produce offspring for this test showed no signs of stress such as high mortality, presence of males or ephippia.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
No post exposure observation period

Test conditions

Hardness:
Total hardness as CaCO3: 180 mg/L
Test temperature:
19-21 °C
pH:
7.9-8.0
Dissolved oxygen:
6.6-11 mg/L
Salinity:
No information
Nominal and measured concentrations:
Nominal concentrations: 1, 2.6, 6.4, 16, 40, 100 µg/L
Measured concentrations (geometric mean): 0.46, 1.1, 3.7, 9.8, 26, 82 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Size, fill volume: 100 ml, 80 ml
- Aeration: no information
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: no information

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water
- Total organic carbon: 1.2 mg/L
- Metals, pesticides: Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice.
- Alkalinity: 88-90 mg/L as CaCO3
- Conductivity: 680-820 µmhos/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: on preparation on each batch of fortified water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light, 8 hours of darkness
- Light intensity: 10-12 µExm-2xs-1

EFFECT PARAMETERS MEASURED: immobilisation of adults, abnormal behaviour, numbers of offspring, number of immobilised offspring, time to first brood release (daily observation)

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100 μg/L, control, solvent (DMF) control
- Results used to determine the conditions for the definitive study: Following 15 days of exposure, 100% and 90% survival was observed among daphnids exposed to the control and solvent control, respectively (pooled control = 95%).
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
82 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 26 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
54 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation

Any other information on results incl. tables

The geometric mean measured concentrations tested, mean survival, reproduction and growth (total body length and dry weight) of parental daphnids (Daphnia magna) during the 21-day static-renewal chronic exposure to the test substance

Geometric Mean

Measured

Concentration (μg/L)

Mean Percent Survival

Mean (SD)a

Number of Offspring Released

Mean (SD)a

Total Body Length (mm)

Mean (SD)a

Dry Body Weight (mg)

Control

90

126 (28)

4.63 (0.12)

0.64 (0.06)

Solvent Control

80

110 (26)

4.61 (0.07)

0.65 (0.07)

Pooled Control

85

118 (28)

4.62 (0.10)

0.65 (0.07)

0.46

100

120 (19)

4.53 (0.11)

0.57 (0.10)

1.1

90

108 (17)

4.56 (0.14)

0.66 (0.11)

3.7

100

115 (20)

4.46 (0.17)b

0.61 (0.09)

9.8

100

110 (15)

4.59 (0.13)

0.59 (0.09)

26

100

99 (19)

4.53 (0.09)

0.64 (0.10)

82

0c

NAd(NA)

NA (NA)

NA (NA)

a: Standard deviation

b: Significantly reduced compared to the pooled control, based on Bonferroni's t-Test. However, due to the lack of statistical significance in the 9.8 and 26μg/L treatment levels, this effect was determined to not be toxicant related.

c: Significantly reduced compared to the pooled control, based on Fisher's Exact Test.

d: NA = Not Applicable. Due to the survival effect observed, this treatment level was excluded from further statistical analysis of reproduction and growth.

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
Based on the most sensitive indicator of toxicity (i.e., survival), the 21-day NOEC for LE 5288 and D. magna was determined to be 26 μg/L. The 21-day LOEC was determined to be 82 μg/L.
The 21-day NOEC for reproduction and growth was 26 μg/L. The 21-day LOEC for reproduction and growth was > 26 μg/L, the highest geometric mean measured concentration statistically analyzed.
The 21-day EC50 value for survival was determined to be 54 μg/L, with 95% confidence intervals of 53 to 54 μg/L.
Since no concentration tested resulted in ≥ 50% reduction in reproduction or growth (total body length and dry weight), the EC50 values for reproduction and growth were empirically estimated to be > 26 μg/L, the highest geometric mean measured concentration statistically analyzed.
Executive summary:

In a full life cycle test (exposure time: 21 days) the toxicity of the test substance towards daphnia magna was assessed in test according to OPPTS Draft Guideline under static renewal conditions. Dimethylformamide was used as a solvent in a concentration of 0.1 mL/L. Test concentrations employed were 1, 2.6, 6.4, 16, 40, 100 µg/L (nominal concentrations) or 0.46, 1.1, 3.7, 9.8, 26, 82 µg/L (measured concentrations; geometrical mean). The endpoints assessed were immobilisation, abnormial behaviour, numbers of offspring, immobilised offspring and time to first brood release and growth.

Based on the most sensitive indicator of toxicity (i.e., survival), the 21-day NOEC for LE 5288 and D. magna was determined to be 26 μg/L. The 21-day LOEC was determined to be 82 μg/L.

The 21-day NOEC for reproduction and growth was 26 μg/L. The 21-day LOEC for reproduction and growth was > 26 μg/L, the highest geometric mean measured concentration statistically analyzed.

The 21-day EC50 value for immobilisation was determined to be 54 μg/L, with 95% confidence intervals of 53 to 54 μg/L. Since no concentration tested resulted in ≥ 50% reduction in reproduction or growth (total body length and dry weight), the EC50 values for reproduction and growth were empirically estimated to be > 26 μg/L, the highest geometric mean measured concentration statistically analyzed.