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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:
March - May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples (50 mL) were taken from the control and each test group from the bulk test preparation at 0 hour and from the pooled replicates at 72 hours for immediate quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Given that the test item was shown to be unstable in the light, additional replicate test vessels were prepared at 0 hours and incubated alongside the test for analysis at 24 and 48 hours.
Vehicle:
no
Details on test solutions:
Information provided by the Sponsor indicated the functional solubility of the test item in water to be less than 320 mg/L. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation, e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
Experimental Preparation
Based on the results of the range-finding test the following test concentrations were selected for the definitive test: 0.32, 1.0, 3.2, 10, 32 and 100% v/v saturated solution. An amount of test item (1000 mg) was dispersed in 2 liters of culture medium with the aid of ultrasonication for 30 minutes. After sonication, any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 32, 10, 3.2, 1.0 and 0.32% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (7.4 mL) to give the nominal test concentrations of 0.32, 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

The stock solutions and each of the prepared test concentrations were inverted several times to ensure adequate mixing and homogeneity.

Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting and all sample bottles were shielded from the light.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 degC.

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 ± 1 degC until the algal cell density was approximately 104 - 105 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 +/- 1 degC
pH:
The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.4 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.

A concentration dependent decline in pH was observed at 0 hours in the range of pH 7.7 at 0.32% v/v saturated solution through to pH 3.2 at 100% v/v saturated solution and did not increase by more 1.5 pH units by 72 hours. The concentration dependent decline in pH was considered to be due to the intrinsically acidic nature of the test item. Following discussion with the Sponsor, no adjustments were made to the pH of the test preparations prior to exposure.

A concentration dependent decline in pH was observed at 0 hours in the range of pH 7.7 at 0.32% v/v saturated solution through to pH 3.2 at 100% v/v saturated solution. This decline was considered to be due to the intrinsically acidic nature of the test item. Following discussion with the Sponsor, no adjustments were made to the pH of the test preparations prior to exposure.
Nominal and measured concentrations:
Nominal concentrations were 0.32, 1.0, 3.2, 10, 32, 100% v/v saturated solution.
Geometric mean measured concentrations were 0.060, 0.14, 0.74, 12, 59 and 226 mg/L.


Time weighted mean measured concentration
0.11, 0.48, 2.0, 14, 60 and 230 mg/L.
Details on test conditions:
Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each 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 characterized by a cell density of 6.05 x 10^5 cells per mL. Inoculation of 900 mL of test medium with 7.4 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 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 (400 – 700 nm) and constantly shaken at approximately 150 rpm for 72 hours to enable algal growth.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 17 - 24 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
A positive control test was conducted October 21, 2013, with used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions for the positive control were similar to those in the definitive test. The 72 hour ErC50 and EyC50 were 1.1 and 0.5mg/L, respectively. The NOEC was 0.25 mg/L and the LOEC was 0.50 mg/L, for both growth rate and yield. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test

Nominal Concentration

(as % v/v saturated solution)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

Biomass Integral

(cells/mL/hour)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

0 – 72 h

% Inhibition

Control

R1

0.072

 

8.57E+05

 

1.28E+07

 

R2

0.071

 

7.97E+05

 

1.20E+07

 

R3

0.073

 

9.39E+05

 

1.42E+07

 

R4

0.073

-

9.60E+05

-

1.42E+07

-

R5

0.072

 

8.77E+05

 

1.32E+07

 

R6

0.072

 

8.63E+05

 

1.30E+07

 

Mean

0.072

 

8.82E+05

 

1.32E+07

 

SD

0.001

 

5.94E+04

 

8.48E+05

 

0.32

R1

0.071

1

8.48E+05

 

1.26E+07

5

R2

0.073

[1]

9.56E+05

 

1.40E+07

[5]

R3

0.073

[1]

9.60E+05

 

1.42E+07

[7]

Mean

0.072

[0]

9.21E+05

[4]

1.36E+07

[2]

SD

0.001

 

6.31E+04

 

8.52E+05

 

1.0

R1

0.068

6

6.66E+05

 

1.02E+07

23

R2

0.068

6

6.70E+05

 

1.01E+07

24

R3

0.072

0

8.62E+05

 

1.28E+07

3

Mean

0.069

4

7.33E+05

17

1.11E+07

17

SD

0.002

 

1.12E+05

 

1.54E+06

 

3.2

R1

0.068

6

6.65E+05

 

1.02E+07

23

R2

0.068

6

6.44E+05

 

9.65E+06

27

R3

0.068

6

6.58E+05

 

1.13E+07

15

Mean

0.068

6

6.56E+05

26

1.04E+07

22

SD

0.000

 

1.04E+04

 

8.44E+05

 

10

R1

0.058

19

3.19E+05

 

5.32E+06

60

R2

0.059

18

3.48E+05

 

5.86E+06

56

R3

0.059

18

3.39E+05

 

5.38E+06

59

Mean

0.059

18

3.35E+05

62

5.52E+06

58

SD

0.001

 

1.47E+04

 

2.96E+05

 

32

R1

0.018

75

1.28E+04

 

2.45E+04

100

R2

0.006

92

3.97E+03

 

-1.58E+05

101

R3

-0.013

118

-1.84E+03

 

-2.24E+05

102

Mean

0.004

95

4.97E+03

99

-1.19E+05

101

SD

0.016

 

7.36E+03

 

1.29E+05

 

100

R1

-0.012

117

-2.27E+03

 

-2.18E+05

102

R2

0.001

99

1.28E+02

 

-1.81E+05

101

R3

-0.003

104

2.37E+02

 

-2.33E+05

102

Mean

-0.005

107

-6.35E+02

100

-2.11E+05

102

SD

0.007

 

1.42E+03

 

2.68E+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
[Increase in growth compared to controls]

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.11, 0.48 and 2.0 mg/L test cultures were observed to be green dispersions. The 14 mg/L test cultures were observed to be pale green dispersions, the 60 mg/L test cultures were observed to be clear colorless solutions whilst the 230 mg/L test cultures were observed to be clear colorless solutions with off-white particles dispersed throughout.

Validity criteria fulfilled:
yes
Conclusions:
The 72 hour EC50 and EC10 for algal growth rate reduction of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione are 20 and 9.4 mg/L respectively. (Envigo Research Limited, 2016).
Executive summary:

In an OECD 201 study, conducted according to GLP, the 72 hour EC50 to freshwater algae (Pseudokirchneriella subcapitata) of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is 20 mg/L (growth rate inhibition). The corresponding 72 hour EC10 for growth rate inhibition is 9.4 mg/L.

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
20 mg/L
EC10 or NOEC for freshwater algae:
9.4 mg/L

Additional information

Key Study:

In an OECD 201 study, conducted according to GLP, the 72 hour EC50 to freshwater algae (Pseudokirchneriella subcapitata) of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is 20 mg/L (growth rate inhibition). The corresponding 72 hour EC10 for growth rate inhibition is 9.4 mg/L (Envigo Research Limited, 2016e).