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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 2015 to 20 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None specified
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing undissolved residue was kept for possible analysis.

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2.0 ml
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Samples were sent to the analytical site on dry ice.
Vehicle:
no
Details on test solutions:
The batch of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] consisted of white to off-white pellets with a purity of 97.8%. The test substance was not completely soluble in test medium at the loading rate initially prepared. The test substance was ground to powder before preparation of test solutions.
Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l applying a two-day period of magnetic stirring. The obtained mixture containing undissolved material was filtered through a 0.45 μm membrane filter (rs55, Whatman) and the filtrate was used as the highest concentrations. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh water algae culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 21 and 22°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).
pH:
The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit)-measured at 7.9 - 8.1
Dissolved oxygen:
Not specified in the study report.
Salinity:
Not applicable; freshwater.
Nominal and measured concentrations:
Samples taken from the limit concentrations were analysed. The actual concentrations were below the limit of quantification of the analytical method already at the start of the test. The lower limit of quantification was 0.025 mg/l.
Details on test conditions:
Test concentrations
2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]: Solutions containing 1.0, 10 and 100% of WSF prepared at a loading rate of 100 mg/l.
Controls: Test medium without test substance or other additives
Replicates: 6 replicates of the control and the limit concentration. 3 replicates of remaining test concentrations. 1 or 2 replicates of each test concentration without algae. 1 replicate of each group for sampling purposes at 24 hour of exposure

Test procedures and conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test solution
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/ml.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 78 to 81 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements
pH: At the beginning and at the end of the test.
The pH of the control solutions should preferably not deviate by more than 1.5 units during the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae.

Recording of cell densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions. In the control and the limit concentrations cell densities were determined at 24 hour intervals. In the remaining groups, only at the end of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.025 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
& yield
Remarks on result:
other: Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l. Concentration relates to this.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
& yield
Remarks on result:
other: Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l. Concentration relates to this.
Details on results:
Measured test substance concentrations
Samples taken from the limit concentrations were analysed. The actual concentrations were below the limit of quantification of the analytical method already at the start of the test. The lower limit of quantification was 0.025 mg/l.

Inhibition of growth rate and inhibition of yield
No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] tested.

The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded the maximum solubility of the test substance in medium.

The 72h-NOEC for both growth rate inhibition and yield inhibition was set at 0.025 mg/l being the limit of quantification of the analytical method and considered the maximum soluble concentration in medium.

Due to the very low solubility of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] in water, concentration levels that might be toxic for algae could not be reached.
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.

The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/l with a 95% confidence interval ranging from 1.2 to 1.5 mg/l. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/l. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.46 mg/l with a 95% confidence interval ranging from 0.43 to 0.49 mg/l. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the 72h-EYC50 for the algal culture tested was within the low end of this range.
Reported statistics and error estimates:
Statistical analysis of the data was not needed as the effects recorded were biologically not significant (<10%).

Percentage inhibition of growth rate (total test period)

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

Mean

Std. Dev.

N

% Inhibition

Control

1.0

10

100

1.778

1.801

1.784

1.788

0.0137

0.0215

0.0184

0.0327

6

3

3

6

 

-1.3

-0.3

-0.6

 

Percentage inhibition of growth rate at different time intervals

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

N

0 – 24 h

24 – 48 h

48 – 72 h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

100

6

6

2.319

2.325

0.0

-0.3

1.607

1.591

0.0

1.0

1.406

1.449

0.0

-3.0

 

Percentage inhibition of yield

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

Mean

Std. Dev.

N

% Inhibition

Control

1.0

10

100

206.1

221.5

209.9

213.6

8.67

14.05

11.47

20.60

6

3

3

6

 

-7.4

-1.8

-3.6

 

Effect parameters

Parameter (mg/l)

NOEC

EC50

Growth rate

Yield

0.0251

0.0251

>0.0251

>0.0251

1Maximum soluble concentration in medium

 

pH levels recorded during the final test

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

pH

t=0h

t=72h

Control

100

8.0

7.9

8.1

8.1

 

Individual cell densities (x104cells/ml)

Time

Replicate

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

Control

1.0

10

100

0h

1

2

3

4

5

6

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

N:

Mean:

Std. Dev.:

CV:

 

6

1.0

0.0

0.0

3

1.0

0.0

0.0

3

1.0

0.0

0.0

6

1.0

0.0

0.0

24h

1

2

3

4

5

6

10.661

9.721

10.611

9.525

10.433

10.097

n.d

n.d.

n.d.

n.d.

n.d.

n.d.

9.865

10.030

10.293

10.185

10.352

10.652

N:

Mean:

Std. Dev.:

CV:

 

6

10.2

0.5

4.7

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

6

10.2

0.3

2.7

48 h

1

2

3

4

5

6

50.515

47.692

53.964

48.696

51.414

53.275

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

47.988

51.014

51.078

42.954

53.131

56.070

N:

Mean:

Std. Dev.:

CV:

 

6

50.8

2.3

4.6

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

6

50.4

4.5

9.0

72 h

1

2

3

4

5

6

199.757

202.774

200.678

209.168

223.140

207.311

229.754

231.364

206.268

216.246

218.726

197.736

192.617

225.944

218.446

186.328

238.619

225.503

N:

Mean:

Std. Dev:

CV:

 

6

207.1

8.7

4.2

3

222.5

14.0

6.3

2

210.9

11.5

5.4

6

214.6

20.6

9.6

n.d. – not determined, n.a. – not applicable

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72h, must be at least 16. In the current test it was found to be 207. The test fulfils this validity criterion.

 

Individual growth rates (day-1)

Time

Replicate

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

Control

1.0

10

100

24h

1

2

3

4

5

6

2.367

2.274

2.362

2.254

2.345

2.312

n.d

n.d.

n.d.

n.d.

n.d.

n.d.

2.289

2.306

2.331

2.321

2.337

2.366

Mean:

Std. Dev.:

N:

CV:

 

2.319

0.047

6

2

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

2.325

0.0266

6

1.1

48 h

1

2

3

4

5

6

1.961

1.932

1.994

1.943

1.97

1.978

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.935

1.966

1.967

1.88

1.986

2.013

Mean:

Std. Dev.:

N:

CV:

 

1.963

0.0228

6

1.2

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

1.958

0.046

6

2.3

72 h

1

2

3

4

5

6

1.766

1.771

1.767

1.781

1.803

1.778

1.812

1.815

1.776

1.792

1.796

1.762

1.754

1.807

1.796

1.743

1.825

1.806

Mean:

Std. Dev:

N:

CV:

 

1.778

0.0137

6

0.8

1.801

0.0215

3

1.2

1.783

0.0184

3

1

1.788

0.0327

6

1.8

n.d. – not determined, n.a. – not applicable

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72h was: 0.8%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control form 0 to 72h, must not exceed 7%. The test fulfils this validity criterion.

 

Section-by-section growth rate

Time

Replicate

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

Control

1.0

10

100

0 – 24 h

1

2

3

4

5

6

2.367

2.274

2.362

2.254

2.345

2.312

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

2.289

2.306

2.331

2.321

2.337

2.366

Mean:

Std. Dev.:

N:

CV:

 

2.319

0.047

6

2

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

2.325

0.0266

6

1.1

24 – 48 h

1

2

3

4

5

6

1.556

1.59

1.626

1.632

1.595

1.644

n.d

n.d.

n.d.

n.d.

n.d.

n.d.

1.582

1.627

1.602

1.439

1.636

1.661

Mean:

Std. Dev.:

N:

CV:

 

1.607

0.0329

6

2.1

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

1.591

0.0792

6

5

48 – 72 h

1

2

3

4

5

6

1.375

1.447

1.313

1.458

1.468

1.378

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.39

1.488

1.453

1.467

1.502

1.392

Mean:

Std. Dev.:

N:

CV:

 

1.406

0.0609

6

4.3

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

1.449

0.048

6

3.3

n.d. – not determined, n.a. – not applicable

The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 27%. According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%. The test fulfils this validity criterion.

 

Individual yields

Time

Replicate

2,2Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

% WSF prepared at 100 mg/l

Control

1.0

10

100

24h

1

2

3

4

5

6

9.7

8.7

9.6

8.5

9.4

9.1

n.d

n.d.

n.d.

n.d.

n.d.

n.d.

8.9

9

9.3

9.2

9.4

9.7

Mean:

Std. Dev.:

N:

CV:

 

9.2

0.47

6

5.2

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

9.2

0.27

6

3

48 h

1

2

3

4

5

6

49.5

46.7

53

47.7

50.4

51.3

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

47

50

50.1

42

52.1

55.1

Mean:

Std. Dev.:

N:

CV:

 

49.8

2.31

6

4.6

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

49.4

4.51

6

9.1

72 h

1

2

3

4

5

6

198.8

201.8

199.7

208.2

222.1

206.3

228.8

230.4

205.3

215.2

217.7

196.7

191.6

224.9

217.4

185.3

237.6

224.5

Mean:

Std. Dev:

N:

CV:

 

206.1

8.67

6

4.2

221.5

14.05

3

6.3

209.9

11.47

3

5.5

213.6

20.6

6

9.6

n.d. – not determined, n.a. – not applicable

 

Overview of % inhibition of growth rate in the reference test:

Nominal conc. K2Cr2O7(mg/l)

Mean

Std. Dev.

N

% Inhibition

Control

0.18

0.32

0.56

1.0

1.8

3.2

1.789

1.758

1.705

1.437

0.994

0.641

0.502

0.0217

0.0229

0.0414

0.0241

0.0430

0.0216

0.0333

3

3

3

3

3

3

3

0.0

1.7

4.7

19.7

44.5

64.2

71.9

 

Overview of % inhibition of yield in the reference test:

Nominal conc. K2Cr2O7(mg/l)

Mean

Std. Dev.

N

% Inhibition

Control

0.18

0.32

0.56

1.0

1.8

3.2

213.756

194.553

166.401

73.570

18.822

5.850

3.530

13.7166

13.4084

20.9357

5.4959

2.6398

0.4397

0.4403

3

3

3

3

3

3

3

0.0

9.0

22.2

65.6

91.2

97.3

98.3

 

Validity criteria fulfilled:
yes
Conclusions:
Due to the very low solubility of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] in water, concentration levels that might be toxic for algae could not be reached.
Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] .

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the OECD series on testing and assessment number 23, 2000.

 

The batch of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] consisted of white to off-white pellets with a purity of 97.8%. The test substance was not completely soluble in test medium at the loading rate initially prepared. The test substance was ground to powder before preparation of test solutions.

 

A Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l and used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

 

Six exponentially growing algal cultures were exposed to an untreated control and the limit concentration, i.e. the undiluted WSF. In addition, three replicates per group were exposed to solutions containing 1.0 and 10% of the WSF in a combined range-finding test. Initial cell density was 104 cells/ml. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

 

Samples taken from the limit concentrations were analysed. The actual concentrations were below the limit of quantification of the analytical method already at the start of the test. The lower limit of quantification was 0.025 mg/l.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Effect parameters obtained in this study are summarized in table below.

Parameter (mg/l)

NOEC

EC50

Growth rate

Yield

0.0251

0.0251

>0.0251

>0.0251

1Maximum soluble concentration in medium

 

Due to the very low solubility of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] in water, concentration levels that might be toxic for algae could not be reached.

Description of key information

Key values determined in a GLP accredited laboratory study in accordance with OECD Guideline 201 and EU Method C.3.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] .

The test substance was not completely soluble in test medium at the loading rate initially prepared. The test substance was ground to powder before preparation of test solutions.

A Water Soluble Fraction (WSF) was prepared at a loading rate of 100 mg/l and used as the highest concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

Six exponentially growing algal cultures were exposed to an untreated control and the limit concentration, i.e. the undiluted WSF. In addition, three replicates per group were exposed to solutions containing 1.0 and 10% of the WSF in a combined range-finding test. Initial cell density was 104 cells/ml. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

The actual concentrations were below the limit of quantification of the analytical method already at the start of the test. The lower limit of quantification was 0.025 mg/l.

 

Effect parameters obtained in this study are summarized in table below.

Parameter (mg/l)

NOEC

EC50

Growth rate

Yield

0.0251

0.0251

>0.0251

>0.0251

1Maximum soluble concentration in medium

 

Due to the very low solubility of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] in water, concentration levels that might be toxic for algae could not be reached.