Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

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:

The study was conducted between 27 April 2010 and 10 June 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

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

Vease comentario más adelante

Principles of method if other than guideline:

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 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)

Remarks:

Certificado en el propio documento

Test material

Test material information

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
CAS number :68188-19-2
Description :pale yellow slightly viscous liquid
Purity :>99%
Batch number :not supplied
Label :Sulfoclor LF 1013 TANQUE 25.09.2009
Date received :5 February 2010
Expiry date :23 September 2010
Storage conditions :room temperature in the dark

The chemical specification of the test item is presented as Appendix 1 please see in attached section.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of test loading rates.

Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
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
The loading rate to be used in the definitive test was determined by a preliminary range-finding test. Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a Water Accommodated Fraction (WAF).
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l.
Amounts of test item (25 and 250 mg) were separately added to the surface of 2.5 litres of reconstituted water to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the reconstituted water 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
In the range-finding test, 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours, the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive test
Based on the results of the range-finding test, a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or adverse reactions to exposure were observed.

Experimental preparation
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the definitive test, the test item was prepared as a Water Accommodated Fraction (WAF).
An amount of test item (400 mg) was added to the surface of 4 litres of reconstituted water to give the 100mg/l loading rate. After the addition of the test item, the reconstituted water 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 approximately 5 hours. The initial instruction was to allow the mixture to stand for 1 hour, however the mixture was allowed to stand for approximately 5 hours in error. This was considered not to affect the outcome of the study as a prolonged standing period would allow settlement of any dispersed globules of test item. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see Appendix 3 in attached section).

Physico-chemical measurements
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

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

Evaluation of data
An estimate of the EL*50 values was given by inspection of the immobilisation data.

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1133) conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at approximately 20°C.

* EL = Effective Loading rate

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:
Water Flea

- Source:
Derived from in-house laboratory cultures.

- Age at study initiation:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

- Feeding during test:
Received no food during exposure

ACCLIMATION
- Acclimation period:
Not stated


- Acclimation conditions:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 degC.


- Type and amount of food:
Each culture was fed daily with a suspension of algae (Chlorella sp.).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation:
No mortality observed


Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.


Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21ºC to 22ºC throughout the test.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
Please see Appendix 4 in any other information on materials and method section.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Please see Appendix 4 in any other information on materials and method section.

Salinity:

freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l.

Details on test conditions:

Exposure conditions
In the definitive test, 250 ml glass jars containing approximately 250 ml of test preparation were used. At the start of the test, 5 daphnids were placed at random in each test and control vessel which contained the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21ºC to 22ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Details on results:

Validation of Mixing Period
Pre-study investigational work (see Appendix 3 in attached section) indicated that there was no significant increase in the measured test concentrations in the WAF by extending the preparation period for longer than 24 hours. Therefore, for the purpose of testing, the test item was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 see in any other information on results.
No immobilisation was observed at the 10 and 100 mg/l loading rate WAF.
Based on this information, a single loading rate of four 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 immobilisation or adverse reactions to exposure were observed.

Definitive Test

Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 see in any other information on result section.
There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation data gave the following results:
Time (h) EL*50 (mg/l Loading Rate WAF)
24 >100
48 >100

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 4 see in any other information on result section. Temperature was maintained at 21ºC to 22ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 3 in any other information on result section).

Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period, the 100 mg/l loading rate was observed to be a clear colourless water column, with small globules of test item floating on the surface. After 23 hours stirring, the 100 mg/l loading rate was observed to be a clear colourless water column, with small globules of test item on the base of the vessel and throughout the water column. After an approximately 5 hour standing period, the 100 mg/l loading rate was observed to be a clear colourless water column, with small globules of test item floating on the surface. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 100 mg/l loading rate was observed to be a clear, colourless solution.

Chemical analysis of test loading rates
Analysis of the test preparations at 0 hours (see Appendix 3 in any other information on result section) showed measured concentrations of 0.381 and 0.388 mg/l. Analysis of the corresponding old media at 48 hours showed measured concentrations of 0.233 and 0.210 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 test water. 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):

Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference item (Harlan Laboratories Ltd Project No: 0039/1133) during the positive control are given in Table 4 see in any other information on result section. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2 see attached section.
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:
Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
24 0.84 0.72 - 0.97
48 0.65 0.58 - 0.72
The No Observed Effect Concentrations after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data, it was not possible to calculate the slope and error of response curve at 48 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.20).

Reported statistics and error estimates:

The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). The EC50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used when two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Any other information on results incl. tables

Table 1              Cumulative Immobilisation Data in the Range-findingTest

Nominal Loading Rate (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
10	0	0
100	0	0

 

Table 2              Cumulative Immobilisation Data in the DefinitiveTest

Nominal Loading Rate (mg/l)		Cumulative ImmobilisedDaphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
100	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

R₁– R₄= Replicates 1 to 4

Table 3              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		100
	*	+	*	+
Height of Water Column (cm)	16.5	16.5	17	17
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

Table 4              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0
0.56	1	1	2	10	3	2	5	25
1.0	7	7	14	70	10	10	20	100
1.8	10	10	20	100	10	10	20	100
3.2	10	10	20	100	10	10	20	100

 

R₁– R₂= Replicates 1 to 2

Pleases also see the attached: Concentration Response Curve after 24 hours in the positive control, Chemical Analysis of Test Loading Rates

, Chemical Specification & Concentration Response Curve after 48 hours in the positive control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of Sulfoclor LF 1013 to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value 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 acute toxicity of Sulfoclor LF 1013 to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of Sulfoclor LF 1013, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21°C to 22ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna was exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.

The 48-Hour EL*₅₀ for Sulfoclor LF 1013 to Daphnia magna based on nominal loading rates was 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 hours showed measured concentrations of 0.381 and 0.388 mg/l. Analysis of the corresponding old media at 48 hours showed measured concentrations of 0.233 and 0.210 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 test water. 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.

The 48-Hour EC₅₀ for the reference item to Daphnia magna based on nominal concentrations was 0.65 mg/l with 95% confidence limits of 0.58 – 0.72 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

 

Conclusion.

The acute toxicity of Sulfoclor LF 1013 to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*₅₀ value 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