Alkanes, C10-13, chloro

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 to 14 October 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Good quality study with good reporting detail; considered adequate for assessment

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Growth of the marine alga (Skeletonema costatum) following exposure to Chlorowax 500C was assessed for up to 10 days.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Alkanes, C10-13, chloro

EC Number:

287-476-5

EC Name:

Alkanes, C10-13, chloro

Cas Number:

85535-84-8

IUPAC Name:

Alkanes, C10-C13, Chloro

Details on test material:

- Name of test material (as cited in study report): Chlorowax 500C
- Substance type: technical product
- Physical state: viscous liquid
- Analytical purity: "100% chlorinated paraffin"
- Impurities (identity and concentrations): free HCl, 7.0 ppm, no stabilisers present
- 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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Radioactivity measurement: At the start of the test, samples (3.0 ml) were taken of each test solution, using the excess remaining after filling the test vessels, and analysed for 14C concentration by liquid scintillation counting. At the end of the test, each blank solution was sampled and analysed in the same manner and 1 ml of one replicate test culture (containing algae) from each concentration was sampled and analysed for 14C concentration by liquid scintillation counting following sample oxidation. On each occasion, samples were analysed from the control and solvent control to ensure that the count lay within 3 standard deviations of the background count for the scintillation counter.

Parent compound measurement: Samples (250 ml) were taken from the controls and each test solution at the start of the test, using the solutions remaining after filling the test vessels, and analysed by a modification to the method of Hollies JI et al. (979). Analytica Chimica Acta 111, 201-213. This involved the extraction of water samples with hexane and then examination by Thin Layer Chromatography.

Test solutions

Vehicle:

yes

Details on test solutions:

The chlorinated paraffin was mixed with 14C-labelled chlorinated undecane by dissolving accurately weighed quantities of both compounds in acetone and mixing known volumes together. Individual stock solutions in acetone were prepared which, when diluted in the algal culture medium, would give the required total chlorinated paraffin concentration. The quantity of 14C labelled chlorinated n-undecane required was calculated such that the radioactivity in the test solutions was approx. twice the background count or between 0.13-0.20 Bq/ml. The proportions of 14C-labelled n-undecane and unlabelled chlorinated paraffin varied according to the total concentration of chlorinated paraffin in the test solution; the amount of 14C labelled material remained approx. the same at different total chlorinated paraffin concentrations.

Test organisms

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: marine alga
- Strain: CCAP 1077/1b
- Source (laboratory, culture collection): laboratory culture
- Age of inoculum (at test initiation): 5 days old
- Method of cultivation: maintained using aseptic techniques to prevent contamination by other microorganisms

ACCLIMATION
- Acclimation period: none specified
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

other: orbital shaking at 80 rpm

Water media type:

saltwater

Limit test:

no

Total exposure duration:

10 d

Post exposure observation period:

None

Test conditions

Hardness:

No data

Test temperature:

Measured daily by thermometer: 19.5-20.6 degrees C, mean 20.3 degrees C
Measured twice/h by automatic temperature recorder: 19.0-20.5 degrees C

pH:

pH of test solutions at the start of the study were 8.38-8.45 and by day 10 were 8.20-8.28

Dissolved oxygen:

No data

Salinity:

30.5 o/oo

Nominal and measured concentrations:

Nominal concentrations: 0, 5.6, 10, 18, 32, 56 and 100 ug/l
Mean measured concentrations: 0, 4.5, 6.7, 12.1, 19.6, 43.1 and 69.8 ug/l
Measured as parent compound in the exposure vessels: 0, 5, 30 and 80 ug/l for the 5.6, 32 and 100 ug/l nominal concentrations respectively
Measured as 14C in the final hexane extracts for parent compound analysis: 0, 3.4, 19.9 and 61.3 ug/l for the 5.6, 32 and 100 ug/l nominal concentrations respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: borosilicate glass conical flasks with polyurethane foam bungs
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml nominal capacity, containing 100 ml test solution
- Initial cells density: nominal value of 0.8 x 10+4 particles/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: Culture medium provided the major nutrient concentrations described by Walsh GE and Alexander SV (1980). Water Air and Soil Pollution 13, 45-55, but contained modified concentrations of trace elements.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater, obtained by continuous pumping from Torbay, Devon. The seawater was filtered to 1 um before use and the complete medium filter sterilised using 0.2 um membrane filters.
- Culture medium different from test medium: No
Analyses of the laboratory seawater for background contaminants are described in a spearate Brixham Laboratory Report.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 14 h light/10 h dark
- Light intensity and quality: continuous illumination of 4000 Lux
- Salinity (for marine algae): 30.5 %o

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: absorbance (optical density) measurements carried out to evaluate the effects of test substance on relative cell density after 4 days and at the end of the test. Electronic particle counts obtained after 2, 3, 4, 7 and 10 days to describe the pattern of growth of the cultures.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.056 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: cell density by absorbance

Remarks on result:

other: 95% CL 0.039-0.113 mg/l; probit slope 3.77

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.043 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: cell density by particle count

Remarks on result:

other: 95% CL 0.027-0.093 mg/l; probit slope 2.41

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.020-0.058 mg/l; probit slope 2.53

Effect concentrations 4

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and cell density

Details on results:

The test substance affected growth during the early stages of the test but the affected cultures ultimately attained cell densities similar to the controls. After 4 days, cell density, as estimated by absorbance, was significantly lower (p=0.01) than in the solvent control at nominal concentrations of 56 ug/l and 100 ug/l (highest tested concentration), at which the reductions compared with the solvent control were 18 and 74% respectively. After 10 days, there were no significant differences between the test substance treatments and the solvent control. Growth rates during the first 2 days were significantly lower (p=0.01) than in the solvent control at nominal concentrations of 32 ug/l and above, but remained lower only for the 100 ug/l treatment during the day 2-3 interval. Growth rates after day 3 were at least as great as in the solvent control for all treatments. See table under "any other information on results section" below.

Reported statistics and error estimates:

The absorbances determined for the control and solvent control on days 4 and 10 were compared using Student's t-test procedures to investigate the possibility of pooling the data for comparisons with the test substance treatments. The absorbances determined on days 4 and 10 were analysed by one-way analysis of variance and Dunnett's procedure was used to compare the test substance treatments with the solvent control. The EC50 and 95% confidence limits were calculated by probit analysis

Any other information on results incl. tables

Mean treatment absorbances and growth rates as a percentage of the corresponding solvent control

 

Nominal concentration (ug/l)	Measured concentration (ug/l). Day 0.	% Solvent control
		Absorbance		Growth rate
		Day 4	Day 10	Days 0-2	Days 2-3
Control	-	104	115	107	99
5.6	4.5	100	104	101	101
10	6.7	104	104	95	102
18	12.1	101	107	88	105
32	19.6	90	108	56*	96
56	43.1	82*	111	53*	97
100	69.8	26*	102	13*	70*

* p = 0.01

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Conclusions:

In a well reported study, the 96h EC50 for cell density for Chlorowax 500C to the marine alga, Skeletonema costatum, was 0.056 and 0.043 mg/l when measured by absorbance and particle count respectively. The 48h EC50 for growth rate was 0.032 mg/l and a 96h NOEC of 0.012 mg/l was obtained.

Executive summary:

In a well reported study, the toxicity of Chlorowax 500C (a C10 -12 chlorinated paraffin; 58% chlorination) to the marine alga, Skeletonema costatum was investigated. Triplicate samples of the 5-day old culture were exposed to nominal concentrations of 0, 5.6, 10, 18, 32, 56 or 100 ug/L (mean measured concentrations of 0, 4.5, 6.7, 12.1, 19.6, 43.1 or 69.8 ug/L) for up to 10 days. Six replicate control and three replicate solvent control cultures were also used. The effects on cell density were assessed by measuring the absorbance and particle count, and the effects on growth rate were also examined.

The test substance affected growth during the early stages of the test but the affected cultures ultimately attained cell densities similar to the controls. After 4 days, cell density, as estimated by absorbance, was significantly lower (p=0.01) than in the solvent control at nominal concentrations of 56 ug/L and 100 ug/L (the highest tested concentration), at which the reductions compared with the solvent control were 18 and 74%, respectively. After 10 days, there were no significant differences between the test substance treatments and the solvent control. Growth rates during the first 2 days were significantly lower (p=0.01) than in the solvent control at nominal concentrations of 32 ug/l and above, but remained lower only for the 100 ug/l treatment during the day 2-3 interval. Growth rates after day 3 were at least as great as in the solvent control for all treatments. The 96h EC50s for cell density, based on measured concentrations, were 0.056 and 0.043 mg/L when measured by absorbance and particle count, respectively. The 48 h EC50 for growth rate was 0.032 mg/L and a 96 h NOEC of 0.012 mg/L was obtained.