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Ecotoxicological information

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:
The study was conducted between 6 May 2010 and 2 July 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Sulfoclor LF 1013 is known to be poorly soluble in water. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several
important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water
Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted
auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period.
Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain
dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the
start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
SULFOCLOR LF 1013
IUPAC Name:
SULFOCLOR LF 1013
Constituent 2
Reference substance name:
Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated
EC Number:
269-145-7
EC Name:
Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated
Cas Number:
68188-19-2
Molecular formula:
CnHxCly(SO2Cl)z n= 14 - 17 or n=17 - 30 ; x=2n+2-y-z
IUPAC Name:
Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated
Details on test material:
Sponsor's identification :SULFOCLOR LF 1013
Description :pale yellow slightly viscous liquid
Batch number :not supplied
Date received :5 February 2010
Expiry date :23 September 2010
Storage conditions :room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Chemical analysis of test loading rates
Samples were taken from the control (replicates R1 - R6 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 - R3 and R4 - R6
pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately 20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.
The method of analysis, stability, recovery and test preparation analyses are described in the attached Appendix 3.

Test solutions

Vehicle:
no
Details on test solutions:
Range-finding test
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test item was prepared as a Water
Accommodated Fraction (WAF).
The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by
exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering twice through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering twice and showed there to be no globules or micro-dispersions of test item present .
An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test, a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron- Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours, the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive test
Based on the result of the range-finding test, a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Experimental Preparation
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/l loading rate. After the addition of the
test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering twice through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100
ml discarded) to give the 100 mg/l loading rate WAF. Microscopic observations of the WAF were performed after filtering twice and showed there to
be no globules or micro-dispersions of test item present.
An aliquot (2 litres) of the WAF was inoculated with algal suspension (57 ml) to give the required test concentration of 100 mg/l loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (please see attached Appendix 3 ).

Physico-chemical measurements
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period.

Evaluation of data
Comparison of growth rates and Comparison of Yield please see in attached section.

Determination of EL*x values
EL*x values were determined by inspection of the growth rate and yield data after 72 hours.

Statistical analysis
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data
after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups.All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
-The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
-The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
-The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test Species
The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from
the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in thelaboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant
aeration and illumination at 21 ± 1ºC.
Prior to the start of the test, sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 104 – 105 cells/ml.
A positive control (Harlan Laboratories Ltd Project Number: 0039/1127) used potassium dichromate as the reference item.
Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. See below.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.
Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH values of the control cultures (see Table 2) were observed to increase from pH 6.8 – 6.9 at 0 hours to pH 8.1 – 8.2 at 72 hours. The pH
deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320pH meter.
Dissolved oxygen:

Not recorded.
Salinity:

freshwater used
Nominal and measured concentrations:
The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
3.3.3 Definitive test
Based on the result of the range-finding test, a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.
Details on test conditions:
Exposure conditions
As in the range-finding test, 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control
and 100 mg/l loading rate WAF treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.40 x 10E5 cells per ml. Inoculation of 2 litres of test medium with 57 ml of this algal suspension gave an initial nominal cell density of 4 x 10E3 cells per ml and had no significant dilution effect on
the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72
hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not stated
Details on results:
RESULTS
Validation of Mixing Period
Pre-study work (see Appendix 3 in attached section) indicated that there was no significant increase in the measured test concentration obtained
from a 100 mg/l loading rate WAF by extending the preparation period for longer than 24 hours.
As such, for the purposes of the test the test item was prepared as a WAF using a 23-Hour stirring period followed by a 1-Hour settlement period.
Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-
finding test are given in Table 1 see in any other information on result section.
The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.
Based on this information, a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 see in any other information on materials and method section. Daily specific growth rates for the control cultures are given in Table 3 see in any other information on result section. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 see in any other information on result section.

The mean cell densities versus time for the definitive test are presented in Figure 1 see in attached section.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 84 after 72 hours. This increase was in line with
the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 4.37 x 10e3 cells per ml
Mean cell density of control at 72 hours : 3.67 x 10e5 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 24% and hence satisfied the validation
criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data
From the data given in Tables 2 see in any other information on materials and method section and 4 see in any other information on result section, it
is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where ErL*x is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P>0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

Inhibition of yield
EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where EyL*x is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out . There were no statistically significant differences (P>0.05) between the control and 100 mg/l loading rate WAF and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and following a 1-Hour settlement period, the WAF was observed to have formed a clear colourless media column with an oily layer of test item at the surface and oily globules visible throughout the media column. Microscopic examination of the WAF showed there to be globules of test item present. As such it was considered appropriate to remove the aqueous phase by filtration through a glass
wool plug twice. Microscopic examination of the WAF after filtration showed there to be no globules or micro-dispersions of test item present.
At the start of the test, all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period, all control and test cultures were observed to be green dispersions.

Physico-chemical measurements
The pH values of each test and control flask are given in Table 2 see in any other information on materials and method section. Temperature was maintained at 24 ± 1ºC throughout the test.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface (see Table 5 in any other information on result section).
Chemical analysis of test loading rates
Analysis of the test preparations at 0 and 72 hours (see Appendix 3 in attached section) showed measured test concentrations to range from 0.22 to 0.34 mg/l.
As Sulfoclor LF 1013 is known to be a complex mixture of sulphochlorinated alkanes, it is likely that one or several of these components will have
dissolved to some extent in the culture medium. The dissolved test item may have been one or several components of the test item. Given that
toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Results with reference substance (positive control):
The positive control was conducted between 12 January 2010 and 15 January 2010.

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h) : 0.49 mg/l*
EyC50 (0 – 72 h) : 0.18 mg/l, 95% confidence limits 0.16 – 0.21 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.


Reported statistics and error estimates:
Statistical analysis
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg Sulfoclor LF 1013/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

4.90E+03

3.40E+05

 

 

 

R2

4.66E+03

4.37E+05

-

-

 

Mean

4.78E+03

3.88E+05

 

 

10

R1

4.15E+03

5.20E+05

 

 

 

R2

4.57E+03

4.03E+05

[7]

[19]

 

Mean

4.36E+03

4.61E+05

 

 

100

R1

5.02E+03

3.17E+05

 

 

 

R2

4.29E+03

3.45E+05

3

15

 

Mean

4.65E+03

3.31E+05

 

 



*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

[Increase in growth compared to controls]

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.079

0.049

0.064

 

R2

0.050

0.083

0.053

 

R3

0.051

0.077

0.057

 

R4

0.049

0.075

0.067

 

R5

0.048

0.079

0.066

 

R6

0.052

0.076

0.055

 

Mean

0.055

0.073

0.060

 

R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg Sulfoclor LF 1013/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.064

 

3.99E+05

 

 

R2

0.062

 

3.38E+05

 

 

R3

0.062

 

3.32E+05

 

 

R4

0.064

-

3.91E+05

-

 

R5

0.064

 

3.99E+05

 

 

R6

0.061

 

3.17E+05

 

 

Mean

0.063

 

3.63E+05

 

 

SD

0.001

 

3.76E+04

 

100

R1

0.064

[2]

3.93E+05

 

 

R2

0.061

3

3.11E+05

 

 

R3

0.059

6

2.81E+05

 

 

R4

0.062

2

3.42E+05

 

 

R5

0.063

0

3.75E+05

 

 

R6

0.060

5

3.01E+05

 

 

Mean

0.062

2

3.34E+05

8

 

SD

0.002

 

4.37E+04

 


*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg Sulfoclor LF 1013/l)

Control

100

*

+

*

+

Height of Media Column (cm)

15

15

15

15

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

 


*= Start of mixing period

+= End of mixing period

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of Sulfoclor LF 1013 on the growth of Desmodesmus subspicatus has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.
Executive summary:

Introduction.

A study was performed to assess the effect of Sulfoclor LF 1013 on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods. 

Following a preliminary range-finding test,Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of Sulfoclor LF 1013, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results. 

Exposure of Desmodesmus subspicatus to Sulfoclor LF 1013 gave EL*50values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 0.22 to 0.34 mg/l.

As Sulfoclor LF 1013 is known to be a complex mixture of sulphochlorinated alkanes, it is likely that one or several of these components will have dissolved to some extent in the culture medium. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.


Conclusion.

The effect of Sulfoclor LF 1013 on the growth of Desmodesmus subspicatushas been investigated and gave EL*50values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

*EL = Effective Loading Rate

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