Calcium 1,4-bis(2-ethylhexyl) bis(2-sulphosuccinate)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 to 30 April 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline compliant GLP study

Justification for type of information:

Data are read-across to a structurally analagous substance (docusate sodium)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: Pseudomonas putida MIGULA
- Method of cultivation: in a glass vial
- Preparation of inoculum for exposure: In the first experiment, the pre-culture had an optical density of 0.302. This pre-culture was adjusted to an optical density of 0.145. In the second experiment the optical density was adjusted from 0.285 to 0.138. The optical density as measured with a Zeiss-Photometer PM 6 and a 1 cm path length.
- Initial biomass concentration: not provided, but the inoculum was diluted 1:10.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16.5 h

Test temperature:

21.4°C for both experiments

pH:

See tables 2 and 3

Duration:

16 h

Dose descriptor:

EC10

Effect conc.:

122 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth inhibition

Duration:

16 h

Dose descriptor:

EC10

Effect conc.:

190 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

16 h

Dose descriptor:

EC50

Effect conc.:

164 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth inhibition

Duration:

16 h

Dose descriptor:

EC50

Effect conc.:

256 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: Since a 5 g/L stock solution was prepared and no insolubility was reported, it is likely that the test concentrations were below the solubility limit in the test medium

Reported statistics and error estimates:

Calculation of the mean optical density per test group and the percent inhibition were performed using the MULTIPLAN software "MIGULA". The ECx values and their corresponding probit values were calculated using the MULTIPLAN software "Programm ZZ". "Programm ZZ" calculates the ECx values by regression analysis of probit transformed % inhibition vs. log concentration.

Table 2 pH-values at the start and end of the exposure of the first experiment

SERWET WH 170	pH	pH
mg/L	start of exposure	end of exposure
0	not provided	not provided
40	6.2	6.8
80	6.2	6.8
160	6.3	6.8
240	6.4	6.8
320	6.6	6.8
400	7.3	6.6

Table 3 pH-values at the start and end of the exposure of the second experiment

SERWET WH 170	pH	pH
mg/L	start of exposure	end of exposure
0	not provided	not provided
40	5.8	6.8
80	5.8	6.8
160	5.9	6.8
240	6.1	6.8
320	6.6	6.8
400	7.1	6.7

Table 4 Optical density (OD) at 436 nm (1cm path length) in the first and second experiment

Note: % inhibition was calculated as: 100*(mean increase of control OD - (mean OD of test culture-initial control OD)/(mean increase of control OD) )

SERWET WH 170	replicate	Experiment 1	Experiment 2
mg/L		initial optical density controls 0.017	initial optical density controls 0.016
0	1	1.705	1.806
	2	1.784	0.706*
	3	1.806	1.796
	4	1.709	1.776
	5	1.745	1.821
	mean	1.750	1.800
	increase of optical density	1.733	1.784
40	1	1.781	1.833
	2	1.786	1.838
	3	1.806	1.813
	mean	1.791	1.828
	% inhibition	-2.4	-1.5
80	1	1.792	1.833
	2	1.806	1.828
	3	1.803	1.821
	mean	1.800	1.827
	% inhibition	-2.9	-1.5
160	1	1.703	1.779
	2	1.681	1.767
	3	1.797	1.775
	mean	1.727	1.774
	% inhibition	1.3	1.5
240	1	1.194	1.329
	2	1.030	1.358
	3	1.254	1.280
	mean	1.159	1.322
	% inhibition	34.1	26.8
320	1	0.183	0.180
	2	0.218	0.183
	3	0.224	0.188
	mean	0.208	0.184
	% inhibition	89.0	90.7
400	1	0.082	0.076
	2	0.075	0.075
	3	0.077	0.085
	mean	0.078	0.079
	% inhibition	96.5	96.5

* not used for calculations

Table 5 ECx-values based on SERWET WH 170 and Docusate Sodium

Experiment	Effect parameter	based on SERWET WH 170	based on Docusate Sodium
		mg/L	mg /L
Experiment 1	EC10	189	121
Experiment 2	EC10	191	123
Mean	EC10	190	122
Experiment 1	EC50	255	164
Experiment 2	EC50	256	164
Mean	EC50	256	164

Validity criteria fulfilled:

yes

Conclusions:

The EC50 as determeined in a Pseudomonas growth inhibition test according to DIN 38412 is 164 mg Docusate Sodium/L.

Executive summary:

The growth inhibition of SERWET WH 170 (which contains 64.2% Docusate Sodium) to Pseudomonas putida MIGULA was determined in a 16 h static GLP Pseudomonas growth inhibition test. The test design was based on the DIN 38412.The nominal test concentrations were 0 (control), 40, 80, 160, 240, 320 and 400 mg SERWET WH 170/L, corresponding to 0 (control), 25.7, 51.4, 102.7, 154.1, 205.4 and 256.8 mg Docusate Sodium/L. Five control replicates and 3 replicates from each test solution were set up. Dose verification analysis was not performed. In order to determine the growth of the cultures, the optical density was measured by photometry at 436 nm. The optical density was determined at 0 hours in the control and about 16 hours after start of the exposure in the control and all test solutions. The EC10 and EC 50 values were calculated by regression of the log transformed concentration vs. probit transformed percent inhibition relative to the average control density. Two experiments were preformed. In the first experiment, the EC10 and EC50 were 121 and 164 mg Docusate Sodium/L, respectively. In the second experiment, the EC10 and EC50 were 123 and 164 mg Docusate Sodium/L, respectively. The mean values for the of the calculated EC10 and EC50 values of both experiments deviated at the maximum of less than 1 % from their mean values and hence, both experiments can be considered to be reliable. The 16 -hour EC10 is 122 mg Docusate Sodium/L and the EC50 is 164 mg Docusate Sodium/L.

This study is considered to be acceptable for the risk assessment.

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

27.01.1992

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Basic data given: comparable to guidelines / standards, meets basic scientific principles, test substance purity not provided.

Justification for type of information:

Data are read-across to a structurally analagous substance (docusate sodium)

Qualifier:

according to guideline

Guideline:

other: DIN 38412-3: Deutsches Einheitsverfahren zur Waser-, Abwasser und Schlammuntersuchung; Testverfahren mit Wasserorganismen (Gruppe L); Bestimmung der Toxizität von Abwässern und Abwasserinhaltsstoffen nach der Dehydrogenaseaktivität mittels 2,3,5- Tripheny

Principles of method if other than guideline:

Toxicity sreening conducted during the OECD 302 B - biodegradation test, Determination of the toxicity based on the dehydrogenase activity via 2,3,5 -triphenyltetrazolium chloride

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge was sampled from the municipal waste water treatment plant Bergisch Gladbach/Refrath (Germany)
- Laboratory culture: no
- Preparation of inoculum for exposure: activated sludge was centrifuged and washed twice and mixed with deionized water : tap water (1:3)
Dry weight= 8.0 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

25 +/- 1°C

Nominal and measured concentrations:

nominal concentrations: 0 (Contol), 100; 200, 500, 1.000, 2.000, 4.000, 6.000 and 8.000 mg Rewopol SB DO 75/L corresponding to 0 (control) , 72, 144, 360, 720, 1.440, 2880, 4.320, 5.760 mg Docusate Sodium/L

Details on test conditions:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water : tap water (1:3)

OTHER TEST CONDITIONS
3 h in the dark at 25 +/- 1°C

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 to 2.5

Reference substance (positive control):

no

Duration:

3 h

Dose descriptor:

EC0

Effect conc.:

144 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: dehydrogenase activity

Duration:

3 h

Dose descriptor:

EC0

Effect conc.:

200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dehydrogenase activity

Details on results:

No further details were provided.

Table 1: Inhibition of dehydrogenase activity:

 

Nominal concentration [mg Rewopol SB DO 75 /L]	Inhibition [%]
Control	-
100	+ 35
200	0
500	20
1000	30
2000	65
4000	85
6000	100
8000	100

 

Validity criteria fulfilled:

yes

Conclusions:

EC0 (dehydrogenase inhibition) = 144 mg Docusate Sodium/L

Executive summary:

The inhibition of the dehydrogenase of sewage treatment plant microorganisms originating from a domestic STP by Rewopol SB DO 75 (which contains equal or more than 72% Docusate Sodium) was determined in a 3 h static non-GLP dehydrogenase inhibition test. The test design was based on the Deutsches Einheitsverfahren (DEV) L 3.The nominal test concentrations were 0 (control), 100, 200, 500, 1000, 2000, 4000, 6000 and 8000 mg Rewopol SB DO 75/L, corresponding to 0 (control), 72, 144, 360, 720, 2880, 4320 and 5760 mg Docusate Sodium/L. Dose verification analysis was not performed. In order to determine the inhibition of the dehydrogenase activity, 2,3,5 -triphenylterazoliumchloride was added to the suspension. Dehydrogenase reduces 2,3,5 -triphenyltetrazoliumchloride to red formazan which is water unsoluble. After 3 hours incubation to test material and 2,3,5 -triphenyltetrazoiumchloride the formazan was extracted using ethanol and the optical density at 480 nm was measured in the supernatant after centrifugation. The EC0 was 144 mg Docusate Sodium/L. Inhibitory effects were observed at 360 mg Docusate Sodium/L and higher.

This study is considered to be acceptable with restrictions for the risk assessment.

Description of key information

No data are available for the registered substance, however data are available for the corresponding sodium salt of the substance, docusate sodium (CAS 577-11-7). These data are used for read-across to the registered substance calcium docusate (CAS 128 -49 -4). The presence of either of the counterions sodium or calcium is not considered to have an impact on the toxicity. It is therefore considered justified to use the data of the sodium salt for read across to the calcium salt of docusate.

EC10 = 122 mg Docusate Sodium/L

Key value for chemical safety assessment

EC50 for microorganisms:

164 mg/L

EC10 or NOEC for microorganisms:

122 mg/L

Additional information

For the data endpoint “Toxicity to microorganisms” in total 2 experimental studies are available: 1 key-study and 1 disregarded study.

The study from Elementis (Schöberl 1993) was classified as Klimisch 1 key study. In this study the growth inhibition of formulated product (which contains 64.2% Docusate Sodium) to Pseudomonas putida MIGULA was determined in a 16 h static GLP Pseudomonas growth inhibition test. The test design was based on the DIN 38412.The nominal test concentrations were 0 (control), 40, 80, 160, 240, 320 and 400 mg formulated product/L, corresponding to 0 (control), 25.7, 51.4, 102.7, 154.1, 205.4 and 256.8 mg Docusate Sodium/L. Five control replicates and 3 replicates from each test solution were set up. Dose verification analysis was not performed. In order to determine the growth of the cultures, the optical density was measured by photometry at 436 nm. The optical density was determined at 0 hours in the control and about 16 hours after start of the exposure in the control and all test solutions. The EC10 and EC 50 values were calculated by regression of the log transformed concentration vs. probit transformed percent inhibition relative to the average control density. Two experiments were preformed. In the first experiment, the EC10 and EC50 were 121 and 164 mg Docusate Sodium/L, respectively. In the second experiment, the EC10 and EC50 were 123 and 164 mg Docusate Sodium/L, respectively. The mean values for the of the calculated EC10 and EC50 values of both experiments deviated at the maximum of less than 1 % from their mean values and hence, both experiments can be considered to be reliable. The 16 -hour EC10 is 122 mg Docusate Sodium/L and the EC50 is 164 mg Docusate Sodium/L. This study is considered to be acceptable for the risk assessment.

In addition to the Klimisch 1 study, there is one Klimisch 3 study for toxicity to microorganisms available. Due to the poor reliability of this study, it is disregarded and not discussed here.

Therefore, the EC10 (122 mg Docusate Sodium/L) and EC50 (164 mg Docusate Sodium/L) reported by Elementis (Schöberl 1993) (Klimisch 1 key study) will be used for the risk assessment.