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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September to 22 December 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported, good quality study to GLP; considered adequate for assessment
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983
Reference Type:
secondary source
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
21 day chronic toxicity (effects on survival, reproduction and growth) assessed following exposure of the freshwater crustracean, Daphnia magna, to Chlorowax 500C (a C10-12 chlorinated paraffin; 58% chlorination)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Chlorowax 500C
- Substance type: technical product
- Physical state: liquid
- Analytical purity: "100% chlorinated paraffin"
- Impurities (identity and concentrations): free HCl, 7 ppm; no stabiliser
- Composition of test material, percentage of components: C10-12 chlorinated paraffin (58% chlorination)
- Lot/batch No.: R201-198
- Expiration date of the lot/batch: "dependent on routine analysis"
- Radiochemical purity (if radiolabelling): >98%
- Specific activity (if radiolabelling): 47 mCi/mmol
- Locations of the label (if radiolabelling): chlorinated n-undecane-6-14C
- Expiration date of radiochemical substance (if radiolabelling): no data
- Stability under test conditions: unstable
- Storage condition of test material: -20 degrees C
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
none

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentration of test substance was measured by 14C analysis. One replicate of each test solution was sampled daily for the first 3 days and at least twice per week thereafter, and analysed by liquid scintillation counting.

Parent compound analysis was also conducted to determine the concentration of the test substance during the first and last weeks. Water samples (250 ml) were extracted with hexane (30 ml) in an upwards displacement liquid/liquid extraction apparatus. Portions of the concentrated extract were examined by thin layer chromatography and by liquid scintillation counting.

Test solutions

Vehicle:
yes
Details on test solutions:
The chlorinated paraffin was mixed with the 14C-labelled chlorinated n-undecane by dissolving accurately weighed quantities of both compounds in acetone and mixing known volumes together. Individual stock solutions were prepared which, when diluted with water, would give the required total chlorinated paraffin concentration. The radioactivity in the test solutions was proportional to the total concentration of chlorinated paraffin. The minimum nominal activity of the solutions was 0.16 Bq/mL. Vehicle was soyabean oil.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: freshwater flea
- Strain/clone: Daphnia magna Straus
- Source: continuous laboratory cultures
- Age of parental stock (mean and range, SD): <24 h old
- Feeding during test: yes
- Food type: suspensions of Chlorella vulgaris and yeast
- Amount: Algae: 4.35 x 10+4 cells/mL on days 0-21; yeast: 1.76 mg/L on days 0-15 and 2.94 mg/L on days 16-21
- Frequency: added continuously to the dilution water

ACCLIMATION
- Acclimation period: none

Study design

Test type:
flow-through
Water media type:
brackish water
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None

Test conditions

Hardness:
mean: 100 mg CaCO3/L, range: 84-121 mg CaCO3/L; analysed at intervals during the study
Test temperature:
nominal range: 20+/-1 degree C; thermometer range: 19.0-20.3 degrees C; automatic recorder range: 19.0-20.7 degrees C; temperature of one replicate of each of the control "channels" measured daily and of each test vessel once/wk by thermometer; in addition, temperature of one control vessel measured by automatic recorder twice/h from day 8 of the test
pH:
range: 6.48-7.65; pH of each test solution measured twice/wk, sampling from alternate replicates
Dissolved oxygen:
range: 5.20-9.30 mg/l; dissolved oxygen concentration of each test solution measured twice/wk, sampling from alternate replicates
Salinity:
No data. To increase the hardness of the freshwater supply, 1% (v/v) seawater was added
Nominal and measured concentrations:
Nominal concentrations: 0, 3.2, 5.6, 10.0, 18.0, 32.0 and 56.0 ug/L
Measured concentration (as measured by 14C analysis): 0, 1.8-4.3, 3.0-6.4, 1.4-9.7*, 12.0-19.0, 23.5-101-4* and 31.9-47.4 ug/L
(* - low or high values omitted from the mean calculation)
Mean measured concentrations: 2.7, 5.0, 8.9, 16.3, 25.5 and 38.7 ug/L

The test concentration by parent compound analysis was also measured in the control, solvent control and all nominal test concentrations. Values were <5, <5, <5, 5, 7.5, 10.80 and 60 ug/L respectively on day 5 and <5, <5, <5, <5, 5 and 7.5 for control, solvent control and nominal test concetrations of 3.2, 5.6 and 10 ug/L respectively on day 21 (the three higher test concetrations were not determined on day 21).
14C analysis in the final hexane extracts during the parent compound analysis gave concentrations for all of the nominal test concentrations of 1.9, 3.3, 5.6, 10.4, 100.3 and 49.1 ug/L respectively on day 5 and 1.7, 2.4 and 1.2 ug/L respectively, with the top three test concentrations not being determined, on day 21.
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): closed - loose fitting lids
- Material, size, headspace, fill volume: borosilicate glass beakers with an overflow positioned such that the vessels contained 1 L of test solution
- Aeration: aerated before use but no further details
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous (dynamic)
- Renewal rate of test solution (frequency/flow rate): flow rate: 25 mL/min of test solution, renewal rate: 3 times/wk the inflowing test solutions were diverted to a second set of test vessels and the surviving parent Daphnia transferred to the new test vessels.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: local town supply water with the addition of 1% (v/v) seawater; freshwater supply was filtered through activated carbon, filtered to 1 um and aerated before use.
- Total organic carbon: range: 0.5-4.0 mg/L, mean: 2.6 mg/L
- Metals: analysed on day 15: cadmium 0.2 ug/L, cobalt: <0.5 ug/L, copper 0.9 ug/L, lead <0.8 ug/L, manganese 0.2 ug/L, nickel 0.4 ug/L, zinc 2.0 ug/L, iron 10.0 ug/L
- Pesticides: analysed for certain organochlorine pesticides but levels described in a separate report and not considered to have affected the validity of the study
- Chlorine: free chlorine <4 ug/L, residual chlorine 7-16 ug/L, mean residual chlorine 11 ug/L
- Alkalinity: 16.0-18.8 mg/L, mean 17.2 mg/L
- Conductivity: 589-761 uS/cm, mean 681 uS/cm
- Culture medium different from test medium: yes - stock cultures maintained in a reconstituted water medium
- Intervals of water quality measurement: Analysed throughout the study at different intervals for hardness, alkalinity, conductivity, total organic carbon, and free and residual chlorine. Analysed once during the study for trace metals.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The initial "parent" Daphnia were examined daily for mortalities and the first release of offspring. Offspring were removed on transfer of the parents to the new test vessels and the number of live and dead offspring counted. At the end of the 21 days, the survivng parent Daphnia were fixed in formaldehyde and the length of each (from the apex of the helmet to the base of the apical spine) was measured.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: control, solvent control, 3.2, 5.6, 10 and 18 ug/L nominal concentrations, semi-static procedure, 14 days
- Results used to determine the conditions for the definitive study: Significant mortality was observed only at 18 ug/L, at which 50% mortality had occurred after 7 days exposure. All animals recorded as immobilised were dead on the following day. Reproduction was reduced by 60% at 18 ug/L when compared with the pooled control and solvent control, and the time to first release of offspring was increased by 2 and 4 days in the two replicates of this concentration compared with all other vessels. Reproduction at the lower test concentrations was not considered to be significantly different. Effects of the test substance were, therefore, seen at a nominal concentration of 18 ug/L (a measured concentration of 10 ug/L) and the no observed effect level was 10 ug/L (a measured concentration of 6.2 ug/L)
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 d
Dose descriptor:
LC50
Effect conc.:
ca. 24 µg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 21-28 ug/L; calculated by probit analysis; based on measured concentrations
Duration:
4 d
Dose descriptor:
LC50
Effect conc.:
ca. 18 µg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 16-20 ug/L; calculated by probit analysis; based on measured concentrations
Duration:
5 d
Dose descriptor:
LC50
Effect conc.:
ca. 14 µg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 12-15 ug/L; calculated by probit analysis; based on measured concentrations
Duration:
6 d
Dose descriptor:
LC50
Effect conc.:
ca. 12 µg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: >99% CL 9-16 ug/L; calculated by the binomial method; based on measured concentrations
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
8.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No CL reported
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: No CL reported
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: No CL reported
Details on results:
- Mortality of parent animals: All the parent Daphnia died within 6 days at mean measured concentrations of 16.3 ug/L and above.
- No. of offspring produced per day per female: No significant effects of the test substance on total young production at measured concentrations of up to 8.9 ug/L - see Table 1 (Under any other information on results) below
- Body length and weight of parent animals: No significant differences in Daphnia length were found between the test substance treatments with surviving animals and the controls or solvent control groups when analysed by one way analysis of variance - see Table 2 (Under any other information on results) below
- Type and number of behavioural abnormalities: Only one animal was recorded as immobilised in addition to the mortalities and therefore immobilisation effects were not analysed separately
- Time to first brood release or time to hatch: Offspring were produced in all vessels of mean measured concentration up to 8.9 ug/L on day 8.
- Other biological observations: 37% of the offspring produced at a measured concentration of 8.9 ug/L were dead when separated from the exposure vessels compared with 6-9% in the control and solvent control groups, and this was found to be significantly greater than in the solvent control (p=0.01). No significant effect was found at test concentrations below this level.
Reported statistics and error estimates:
The median lethal concentrations and 95% confidence limits were calculated for 3, 4 and 5 days by probit analysis using a computer programme and the 6 day LC50 and confidence limits was calcuated by the binomial method.
The lengths of the surviving Daphnia were analysed by one way analysis of variance.

Any other information on results incl. tables

Table 1: no. of offspring produced/female

Nominal concentration (ug/L)

Mean total no of offspring/parent

Dead offspring/parent

% dead offspring

Control 1

50.5

4.3

8.5

Control 2

105.4

8.2

7.8

Solvent control

100.9

6.3

6.2

3.2

56.6

3.2

5.7

5.6

87.8

8.7

9.9

10.0

50.6

18.5

36.6

 

Table 2: Lengths of surviving parent Daphnia after 21 days

Nominal concentration (ug/l)

Means (micrometer eyepiece scale divisions)*

Mean lengths (mm)*

Control 1

7.24

4.53

Control 2

7.28

4.55

Solvent control

7.26

4.54

3.2

7.22

4.51

5.6

7.36

4.60

10.0

7.19

4.49

*Calibration of scale: Divisions x 0.625 = mm

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
In a well conducted, GLP study, the 21-day NOEC for Chlorowax 500C (a C10-12 chlorinated paraffin; 58% chlorination) to the freshwater crustracean, Daphnia magna, was 5.0 ug/L (mean measured concentration). A significant reduction in the viability of the offspring occurred at a measured concentration of 8.9 ug/L.
Executive summary:

In a well conducted GLP study, the 21-day toxicity of Chlorowax 500C (a C10-12 chlorinated paraffin; 58% chlorination) to the freshwater crustracean, Daphnia magna, was investigated using a continuous flow procedure (in brackish water). Replicate samples of 10 Daphnia/group, aged <24 h, were exposed to nominal concentrations of 3.2, 5.6, 10, 18, 32 and 56 ug/L (mean measured concentrations of 2.7, 5.0, 8.9, 16.3, 25.5 and 38.7 ug/L) for 21 days. The animals were fed throughout exposure and the effects on survival, reproduction and growth were assessed.

All parent Daphnia exposed to measured concentrations of 16.3 ug/L and above died within 6 days. Only one animal was recorded as immobilised in addition to the mortalities and therefore immobilisation effects were not analysed separately. In the surviving groups, there was no significant effects on parental length or on total young production. Offspring were produced in all vessels by day 8, with a significantly greater number of those produced at a measured concentration of 8.9 ug/L being found dead when compared with the solvent controls. There were no significant effects on survival, reproduction and growth (length after 21 days) for Daphnia exposed to a measured concentration of up to 5 ug/L. Therefore, the NOEC for Chlorowax 500C under these conditions was 5 ug/L and the LOEC was 8.9 ug/L. The "maximum acceptable toxicant concentration" (MATC) was, therefore, between 5.0 and 8.9 ug/L, the geometric mean being 6.7 ug/L.