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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
REG (EC) No. 440/2008, May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Qualifier:
according to
Guideline:
other: Chemical Substance Control Law 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of Japanese METI, MHLW and MOE
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 100 , 50, 25, 12.5, 6.25 mg/L as WAF and control
- Sampling intervals: start and end of each renewal period
- Sampling method: samples were taken from flasks containing daphnids
- Sample storage conditions before analysis: processed (freeze-dried) samples were analysed on the day of sampling
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAFs)
Supersaturated solutions were prepared by dispersing/dissolving the test item into the test medium (ISO Medium) at nominal concentrations of 100, 50, 25 and 12.5 mg/L. These dispersions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at the test temperature. The resulting dispersions were opalescent. The non-dissolved test material was removed by filtration through a 0.22-µm filter to provide the WAFs. The filtrates were clear and transparent. The lowest nominal test concentration of 6.25 mg/L was prepared by diluting the 100 mg/L WAF.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: National Institute of Public Health, 1097 Budapest Gyáli u. 2-6. Hungary. The Daphnia are bred in-house.
- Age at study initiation: < 24 h
- Feeding during test: none

ACCLIMATION
None; the same medium (ISO medium according to the OECD and EC guidelines) was used for breeding and tests. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behavior or significant decrease of population is recorded.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
249 mg/L as CaCO3 (calculated)
Test temperature:
20.1-21.1 °C
pH:
7.69-7.91
Dissolved oxygen:
8.1-8.8 mg O2/L
Nominal and measured concentrations:
nominal loading rates: 100, 50, 25, 12.5, and 6.25 mg/L as WAF
first renewal period (0-24 h):
freshly prepared test media: 2.95, 1.91, 1.10, 0.35 and 0.16 mg/L
aged test media (24 h): 2.48, 1.13, 0.46, 0.23, and 0.09 mg/L

second renewal period (24-48 h):
freshly prepared test media: 3.13, 2.18, 2.15, 1.51 and 0.34 mg/L
aged test media (24 h): 2.28, 2.30, 1.11, 1.02 mg/L and geometric mean measured concentrations: 2.69, 1.81, 1.05, 0.59 and 0.12 mg/L

biological results are based on the nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: open glass beakers containing approx. 25 mL medium
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: at least 5 mL medium per animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: water temperature, oxygen concentration and pH in the controls and the test solutions were measured at the beginning and at the end of each renewal period.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light and 8-hour dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : number of immobilised animals and percentage of immobility at 24 and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: nominal 100, 10, 1, and 0.1 mg/L
- Results used to determine the conditions for the definitive study: 100% immobility after 48 hours at the nominal loading rate of 100 mg/L; no immobility at the lower test concentrations
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 33-42 mg/L; results are expressed in terms of loading rate
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: results are expressed in terms of loading rate
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: results are expressed in terms of loading rate
Details on results:
- Number and percentage of immobiliosed daphnids: see Table 1
- Behavioural abnormalities: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 24-hour EC50: 0.98 mg/L (95% confidence limits. 0.92-1.06 mg/L)
Value is within the range of the inter laboratory ring test data.
Reported statistics and error estimates:
The 24- and 48-hour EL50 values with 95 % confidence limits were determined by Probit analysis using SPSS/PC+ and TOXSTAT software, respectively.
The 48-hour EL100 value was determined directly from the raw data.
For the determination of the LOELR and NOEL, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Table 1: Number and percentage of immobilised daphnids

Nominal loading rate (mg/L)

Cumulative number of immobile Daphnia

24 hours

48 hours

R1

R2

R3

R4

total

%

R1

R2

R3

R4

total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

6.25

0

0

0

0

0

0

0

0

0

0

0

0

12.5

0

0

0

0

0

0

1

1

1

2

5

25

25

0

0

0

0

0

0

3

2

1

2

8

40

50

0

0

0

0

0

0

3

0

1

2

6

30

100

2

2

4

2

10

50

5

5

5

5

20

100

R: replicate number, 5 daphnids per replicate

Validity criteria fulfilled:
yes
Remarks:
In the control, no Daphnia have been immobilised or trapped at the surface of the water. The dissolved oxygen concentration at the end of the test was > 3 mg/L in control and test vessels.
Conclusions:
The test material is toxic to daphnia with a 48-hour EL50 between 10 and 100 mg/L (calculated value: 37 mg/L).

Description of key information

48-h EC50 37 mg/L for Daphnia magna (based on nominal loading rates) (OECD 202, EU C.2)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
37 mg/L

Additional information