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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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Remarks:
Study conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
no
Remarks:
Study conducted according to guideline in effect at time of study conduct
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 20.0% solids in water

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations Sampled: All test samples were analysed.
- Sampling Times: One sample plus a back-up sample from each test concentration, including the dilution water control, before and after addition of the diet solution, on day 0 and day 2 of the test before the test solutions were poured into the replicate test chambers. One sample plus a back-up sample from each test concentration, including the dilution water control, after addition of the diet solution, on days 9, 16, and 19 of the test before the test solutions were poured into the replicate test chambers. One sample plus a back-up sample from 2 of the 10 replicate chambers at each test concentration, including the dilution water control, on days 5, 12, 19 and 21 of the test. On day 19 test samples for the 0.63 mg total solids/L dose level from 2 chambers due to total adult immobility and on day 21 test samples for the 0.63 mg total solids/L and the 2.5 mg total solids/L dose level from 4 chambers due to total adult immobility.
- Sample storage conditions before analysis: Samples and back-up samples were stored refrigerated when not in use.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
On day 0 and on each renewal day (Monday, Wednesday, and Friday), a primary stock solution of 75.0 mg/L was prepared by adding 150 milligrams of the test substance and bringing this up to 2000 mL in test facility well water in a 4-L Erlenmeyer flask and stirring for approximately 1 hour for the day 0 stock solution and approximately 2 hours for all subsequent primary stock solutions prepared during the study. The stock solutions were slightly cloudy with surface film and undissolved test substance. Final nominal test concentrations of 3.15, 6.5, 12.5, 25, and 50 mg/L test substance (which correspond to nominal total solids concentrations of 0.63, 1.3, 2.5, 5.0, and 10 mg total solids/L) were prepared by diluting appropriate amounts of the primary stock solution with test facility well water. At test start and at each renewal, each test concentration and the dilution water control received 2.1 mL/L of algae (final algal concentration of approximately 62,500 cells/mL in test solutions) and 3 mL/L of yeast, cereal leaves and trout chow mixture (YCT). Final test solutions were stirred for approximately 7-10 minutes before use. At test start, the test facility well water and nominal 0.63 mg/L test solutions were cloudy and pale green in colour (due to the presence of algae) with visible particles of YCT. All other test solutions were cloudy and pale green with particles of YCT, a precipitate and surface film was also present.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea.
- Source: Reared at the performing lab.
- Age of parental stock (mean and range, SD): Neonates used in this test were less than 24 hours old and were collected from the 5th brood of 15-day old parent daphnids.
- Feeding during test: Yes.
- Food type and amount: Daphnids were fed on a daily basis with 3 mL/L of a yeast, cereal leaves and trout chow (YCT) mixture and the green alga, Pseudokirchneriella subcapitata, at a rate of approximately 62,500 cells/mL of culture media. The YCT mixture was standardized to 1700-2100 mg/L solids. The combination of YCT and alga is equivalent to approximately 0.1-0.2 mg total organic carbon per daphnid per day.
- Frequency: Daily

ACCLIMATION
- Daphnia magna were reared at the testing facility in 1000-mL glass beakers (8-10 per beaker) which contained 1000 mL of aerated, filtered testing facility well water and held at approximately 20.0°C.
- Type and amount of food: A combination of YCT (yeast, cereal leaves and trout chow mixture) and alga, equivalent to approximately 0.1-0.2 mg total organic carbon per daphnid per day. Algal feeding was 62,500 cells per mL of medium. The YCT mixture was standardized to 1700-2100 mg/L solids.
- Feeding frequency: Daily, excluding weekends and holidays.
- Health during acclimation (any mortality observed): No adult immobility was seen during the 48-hour pre-test period in the culture used to supply neonates for testing.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d

Test conditions

Hardness:
Total alkalinity, EDTA hardness, and conductivity values for the dilution water control throughout the study ranged from 75-94 mg/L as CaCO3, 116-178 mg/L as CaCO3, and 201-332 µmhos/cm, respectively. Total alkalinity, EDTA hardness, and conductivity values for the highest test concentration with surviving organisms (nominal 50 mg/L test substance) throughout the study ranged from 83-100, 102-213, and 206-351, respectively.
Test temperature:
between 20.3 and 21.7°C
pH:
The pH ranged from 8.0 to 8.3 in new solutions and from 7.9 to 8.4 in old solutions but did not deviate by more than 0.5 units over a 48- or 72-hour period between renewals within the same replicate at all concentration levels.
Dissolved oxygen:
8.3 mg/L or 91% of saturation (based on an air saturation value of 9.1 mg/L at 20°C
Nominal and measured concentrations:
Nominal: 0, 3.15, 6.5, 12.5, 25, and 50 mg/L
Corrected Nominal Solids: 0, 0.63, 1.3, 2.5, 5.0, and 10 mg total solids/L
Measured: 0, 0.439, 0.826, 1.66, 3.49, and 6.59 mg total solids/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (250 mL) containing 100 mL of test solution (3.3 cm depth)
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed every Monday, Wednesday, and Friday during the study.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

OTHER TEST CONDITIONS
- Adjustment of pH: No data
- Photoperiod and light intensity: A photoperiod of 16 hours light (approximately 226-368 Lux) and 8 hours darkness was employed, which included approximately 30 minutes of transitional light (approximately 11-35 Lux) between the light and dark intervals.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations were made daily on the number of surviving adult daphnids, occurrence of sublethal effects, and production of live or immobile young. The criterion for immobility was the lack of response to application of a gentle stimulus. Immobile daphnids (adults and young) were discarded on renewal days. Live or immobile young were counted and removed on renewal days. Length and dry weight of surviving adult daphnids were determined at test end.

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
6.59 other: mg total solids/L
Conc. based on:
other: Mean measured concentrations
Basis for effect:
other: all endpoints
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 6.59 other: mg total solids/L
Conc. based on:
other: Mean measured concentrations
Basis for effect:
other: all endpoints
Duration:
21 d
Dose descriptor:
other: MATC (maximum-acceptable-toxicant concentration)
Effect conc.:
> 6.59 other: mg total solids/L
Conc. based on:
other: Mean measured concentrations
Basis for effect:
other: all endpoints
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 6.59 other: mg total solids/L
Conc. based on:
other: Mean measured concentrations
Details on results:
See Table 1.
There was a significant increasing trend in the total number of live young produced and in the total number of live young per surviving adult. Since an increase in young is not considered adverse, the NOEC for these two responses was determined to be the highest test concentration.

Any other information on results incl. tables

Table 1 

 

Mean (Standard Deviation)

Mean, Measured Concentrations (mg/L)

Percent Adult Survivala

First Reproduction Dayb

Total Live Youngc

Total Immobile Youngd

Adult Length (mm)e

Adult Dry Weight (mg)e

Dilution Water Control

80

8.5 (1.1)

101.8 (22.0)

0.0 (0.0)

4.2 (0.2)

0.6 (0.3)

0.439

60

8.6 (2.1)

82.0 (52.0)

0.0 (0.0)

4.1 (0.3)

0.4 (0.3)

0.826

60

8.2 (0.6)

145.3 (13.5)

0.0 (0.0)

4.4 (0.2)

0.5 (0.1)

1.66

50

8.2 (1.1)

150.2 (42.5)

0.0 (0.0)

4.3 (0.3)

0.5 (0.1)

3.49

90

7.8 (0.4)

138.0 (24.6)

0.0 (0.0)

4.3 (0.3)

0.5 (0.2)

6.59

80

8.0 (0.7)

147.1 (34.1)

0.0 (0.0)

4.2 (0.4)

0.5 (0.1)

a    Percent of adult daphnids alive at the end of the test

b    First day that reproduction was observed

c    Sum of live young produced per surviving adult in 21 days

d    Sum of immobile young produced per surviving adult in 21 days

e    Per surviving adult

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Based on mean measured concentrations and all endpoints at the end of the study:
21-day NOEC (no-observed-effect concentration) = 6.59 mg total solids/L
MATC (maximum-acceptable-toxicant concentration) >6.59 mg/L
LOEC (lowest-observed-effect concentration) >6.59 mg/L
EC50 for adult survival >6.59 mg/L
Executive summary:

In a 21-day chronic, static renewal study with Daphnia magna, a total of 10 replicates, each containing one, 24 hour-old neonate, were tested per concentration (10 neonates/concentration) and dilution water control. Nominal test substance concentrations were 0, 3.15, 6.5, 12.5, 25, and 50 mg/L. Corrected nominal solids concentrations were 0, 0.63, 1.3, 2.5, 5.0, and 10 mg total solids/L. Measured test substance concentrations were 0, 0.439, 0.826, 1.66, 3.49, and 6.59 mg total solids/L. The daphnids were fed Pseudokirchneriella subcapitata at a rate of approximately 62,500 cells/mL of test solution and 3 mL/L of a yeast, cereal leaves and trout chow mixture (YCT) on a daily basis. The combination of alga and YCT was equivalent to approximately 0.1-0.2 mg total organic carbon (TOC) per daphnid. Test solutions (old and new) were maintained between 20.3 and 21.7°C. Observations of immobilization, sublethal effects, and production of young were made daily. Length and dry weight of surviving adult daphnids were determined at test end. The 21-day NOEC (no-observed-effect concentration) was 6.59 mg total solids/L, based on mean measured test concentrations and all endpoints at the end of the study. The MATC (maximum-acceptable-toxicant concentration), and LOEC (lowest-observed-effect concentration) were >6.59 and >6.59 mg/L, respectively. The EC50 for adult survival was greater than >6.59 mg/L.