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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 January 2002 - 19 March 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Restriction mainly due to the lack of analytical confirmation of test material concentrations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(testing on Water Accommodated Fractions. Some minor deviations from protocol)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):
- Physical state: Liquid
- Lot/batch No.: NDA
- Expiration date of the lot/batch: August 06
- Storage condition of test material: Room temperature
- Other:

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Samples were taken from each water-acommodated fraction (WAF) and control solution on Day 0. Samples were analysed for Total Organic Carbon (TOC) content.

Test solutions

Vehicle:
no
Details on test solutions:
Due to the complex nature of the test substance, samples of the WAFs were analyzed for Total Organic Carbon (TOC) content. The reported TOC values represent the average of four 5mL injections. The following results of TOC testing were recorded for 'Day 0' samples:

LOADING RATE (mg/L) TOC (ppm C)

0 0.4428
1 0.5346
3 0.8905
10.5 1.0726
30.5 2.0138
99.5 5.1534

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnids were cultured in-house at the test facility. The original culture was supplied by Aquatic Biosystems, Inc., Fort Collins, CO.

Daphnids were kept in 1-litre glass culture vessels with reconstituted water (at 20 ± 2°C) supplemented with vitamin B12 and selenium. Cultures of Daphnia were fed 1 mL of 1.3 x 108 cells/mL Pseudokirchneriella subcapitata and 4.8 mL of yeast / salmon starter / wheat grass (YTC) mixture daily. Both algae and YTC were supplied by Aquatic Biosystems, Inc., Fort Collins, CO.

The juvenile daphnids used in the test were produced from the laboratory in-house cultures and were less than 24-hours old.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None

Test conditions

Hardness:
92-96mg/l CaCO3
Test temperature:
19.0-20.2 C
pH:
7.6-7.7
Dissolved oxygen:
8.1-8.3 mg/l
Salinity:
Na/K ratio 5.9 : 1
Ca/Mg ratio 2.8 : 1
Nominal and measured concentrations:
0, 1, 3, 10.5, 30.5, 99.5 mg/l (loading rates)
Details on test conditions:
Test System:
Water Accommodated Fractions (WAFs). Individual treatment WAFs were prepared by adding the test substance to dilution water in glass aspirator bottles and removing the WAF of each mixture for testing rather than prepare dilutions of a stock solution. The test substance was delivered through a plastic syringe on to the surface of the water in the aspirator bottle. The syringe was weighed before and after adding the test substance to determine the actual loading level. The mixing vessels were sealed with Teflon® covered rubber stoppers. The mixtures were stirred with a vortex ≤ 10% static liquid depth for approximately 24 hours on magnetic stirplates with Teflon® coated stirbars at room temperature (22°C ± 2°C). After stirring, the mixtures were allowed to settle for 1 hour, before the aqueous portions were removed through the outlet at the bottom of the vessels. Four replicate test chambers were prepared for each treatment by completely filling the chambers with the WAF (no headspace). Four replicates of the control were prepared in the same manner using laboratory dilution water. All test chambers were sealed with ground glass stoppers immediately to avoid loss of volatile components. During mixing, the treatments appeared clear and colourless with test substance floating on the water surfaces.

Test chambers:
125 mL glass Erlenmeyer flasks containing approximately 140 mL of solution (no headspace).

Number of organisms:
120. Five per test chamber.

Organism selection:
Organisms were randomly assigned to intermediate chambers using a computer generated randomization schedule and then transferred to their respective test chambers. The test chambers were randomly positioned within the test area.

Neonates from parents with ≤20% mortality were selected. Neonates were selected from a pool of organisms larger than that needed for the study. The study director or his designee determined organism suitability.

Lighting:
Approximately 16 hours light and 8 hours dark per day. Daylight intensity ranged from approximately 699 to 686 Lux during full daylight periods of the study

Feeding:
Test organisms were not fed during the study.

Test concentrations:
Test concentrations used in the definitive test were based on the results of a preliminary limit test.

No range finding study was performed with this study. A limit test was conducted at a nominal loading level of 100 mg/L. In general, the tests were performed by using the same experimental procedures as the definitive tests. The limit test resulted in no mortality at 24 hours and 100% mortality at 48 hours.
Reference substance (positive control):
yes
Remarks:
Potassium chloride

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EL50
Remarks:
Loading rates
Effect conc.:
ca. 91.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval of 51.3 mg/L to >99.5 mg/L
Details on results:

% immobilisation

Actual
Loading
(mg/L) 24 hours 48 hours
0 (Control) 0 0
1 0 0
3 0 0
10.5 0 0
30.5 0 30
99.5 0 50

Results with reference substance (positive control):
The 48-hour EC50 for the reference substance (KCl) run during the test was 670 mg/L (no confidence interval could be calculated). This result corresponds with values obtained from the U.S. EPA Ecotoxicology Database System and with historical testing data found in the Daphnia Reference Toxicity logbook.
Reported statistics and error estimates:
The 48-hour EL50 value was calculated using the Probit analysis procedure of SAS.

Any other information on results incl. tables

None of the control Daphnia were immobilized or trapped at the surface of the water. Some surviving daphnids were observed as lethargic.

% of surviving daphnids observed as lethargic

Actual
Loading
(mg/L)

24 hours

48 hours

0

0

0

1

0

0

3

0

15

10.5

0

10

30.5

0

43

99.5

5

50

Dissolved oxygen remained > 60% of the air saturation value.  The pH did not vary more than 1 unit.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this acute daphnia toxicity study of the test substance, the 48-hour EL50 was 91.4 mg/L (with a 95% confidence interval of 51.3 mg/L to >99.5 mg/L).
Executive summary:

The acute aquatic toxicity potential of the test substance was evaluated in a 48-hour static Daphnia magna test. The GLP test was conducted largely in accordance with OECD Test Guideline 202. Due to the nature of the test substance, test solutions were prepared as Water Accomodated Fractions (WAFs). Based on the results of a preliminary limit test in which a nominal loading rate of 100 mg/L resulted in no mortality at 24 hours, but 100% mortality at 48 hours, the definitive test used nominal loading rates of 0, 1, 3, 10, 30 and 100 mg/L. The 48-hour EL50 (immobilisation) was calculated to be 91.4 mg/L (95% CI: 51.3 - >99.5mg/L). The maximum loading rate causing no immobilisation was 10.5 mg/L.