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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
None.
Analytical monitoring:
yes
Details on sampling:
Range-finder and Definitive tests

Experiments with both fresh and aged flasks started at the same time with the same inoculum and the same control group. At 0h, cell concentration was quantified in the culture stock solution. After 24 and 48h of incubation, aliquots of 200 µL were sampled from each inoculated test flask with a pipet and transferred into a microplate. At 72h, the samples with high cell density were dilute to ¼ (50 µL + 150 µL of filtered dilution water) before transfer into the microplate. Microplates were then analysed using a flow cytometer (Guava easyCyte™ flow cytometer Merck Millipore). Time between sampling and measurement wais approximately 15 – 30 min.
Test item concentrations were measured at 0 and 72 hours in all flasks (fresh and aged). In addition, test item concentrations were also measured in the aged flasks prior to the aging process (exposure to light), in order to check consistency between nominal and actual concentrations of test item.
Vehicle:
no
Details on test solutions:
Test item solubility is 320 mg/L and does show any difficulty to solubilize. Therefore, stock solution preparation consisted in mixing 100 mg of test item with 1L algae medium for 45 minutes at high speed stirring at room temperature. Aged stock solution was prepared 3 days before fresh stock solution. During these 3 days, the aged stock solution was exposed to OECD 201 experimental lighting conditions to totally degrade the test item in its degradation product.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK). Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline. The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.

Three days before the start of the exposure, two pre-cultures were prepared by inoculating each stock suspension of algae into sterile dilution water. The pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only used if the first one was damaged.

At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 10 000 cells/mL as recommended in the OECD 201 guideline.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None.
Post exposure observation period:
None.
Hardness:
Not measured.
Test temperature:
22.3°C - 22.9°C (min - max)
pH:
8.1 - 9.4 (min - max)
Dissolved oxygen:
4.9 - 20 mg/L (min - max) (high levels of dissolved oxygen were due to O2 algal production in a sealed system.
Salinity:
Freshwater.
Conductivity:
Not measured
Nominal and measured concentrations:
Range finder
Aged solution nominal concentrations: 0 ; 1 ; 10 and 100 mg/L
Aged solutions measured concentrations at 0h: 0 ; 0 ; 0.07 and 0.34 mg/L
Aged solutions measured concentrations at 72h: 0 ; 0 ; 0 and 0.12 mg/L

Fresh solution nominal concentrations: 0 ; 1 ; 10 and 100 mg/L
Fresh solutions measured concentrations at 0h: 0 ; 1 ; 9.9 and 99 mg/L
Fresh solutions measured concentrations at 72h: 0 ; 0 ; 0.02 and 5.18 mg/L

Definitive test
Aged solution nominal concentrations: 0 ; 0.032 ; 0.1 ; 0.32 ; 1 and 3.2 mg/L
Aged solutions measured concentrations at 0h: 0 ; 0 ; 0 ; 0 ; 0 and 0 mg/L
Aged solutions measured concentrations at 72h: 0 ; 0 ; 0 ; 0 ; 0 and 0 mg/L

Fresh solution nominal concentrations: 0 ; 0.032 ; 0.1 ; 0.32 ; 1 and 3.2 mg/L
Fresh solutions measured concentrations at 0h: 0 ; 0.03 ; 0.10 ; 0.32 ; 1.0 ; 3.2 mg/L
Fresh solutions measured concentrations at 72h: 0 ; 0 ; 0 ; 0 ; 0 and 0 mg/L
Details on test conditions:
The substance is known to be unstable in the test conditions (photolysis was hypothesized and shown, as described below). As the volatility of the potential metabolites was unknown, all experiments were carried out in a way that prevents metabolites evaporation (glass bottles tightly closed with bungs and without headspace were used). Since photolysis was expected, all vessels used in every trials were always remained identical, as flask characteristics may affect light composition and thus photodegradation rate.

STABILITY TEST
A series of four test item concentrations was prepared (0, 1, 10 and 100 mg/L) from successive dilutions of a stock solution: a known volume of test item (100.1 mg) was poured into a volumetric flask, the volume was then made up to 1000 mL with the test medium. The solution was kept under high speed stirring at ambient temperature during approximately 45 min. with a magnetic stir bar before dilution. Four abiotic vessels were prepared at each test concentration. Two vessels were incubated under the same environmental conditions as the algae definitive test and two were similarly incubated in the dark under the same other environmental conditions as the definitive test (exact same flask, same temperature and same agitation method). The test item concentrations were determined in each test vessel at T0 and every 24h until 72h.

RANGE-FINDING TEST
As the substance was proven to be rapidly degraded by light in the stability test, it was hypothesized that the degradation products (not identified) may be as toxic as the parent compound (tetramethylthiuram monosulfide). To verify this hypothesis, additional flasks pre-exposed to light for a sufficient time to obtain 100% degradation products (thereafter called “aged flasks”) were inoculated and incubated under the same conditions as the "fresh flask". To do so, a first test item stock solutions (at 100 mg/L nominal concentration) was prepared. A known mass of test item (100.1 mg) was poured into a volumetric flask. The flask was then filled up to 1000 mL QSP with the test medium. The solution was kept under high speed stirring at ambient temperature during approximately 45 min. with a magnetic stir bar. Then it was incubated in the phytoculture cabinet within test conditions (temperature and light intensity) during 72 hours (i.e. until start of the test).

At the beginning of the test, a second test item stock solution was similarly prepared for the "fresh flasks". Algae were exposed to a serie of test item concentrations (1, 10 and 100 mg/L of nominal concentrations) prepared from successive dilutions of both fresh and old stock solutions. An inoculated control flask (labelled T) was prepared and incubated under the same conditions, with no test item. Three vessels were prepared at each test concentration and six vessels for the control group; each vessel was inoculated with ca 10 000 cells/mL.

Test item concentrations in the pre-exposed flasks were the same as in the series of non-pre-exposed flasks (fresh flasks) and performed in triplicate as well. During this range-finding test, test item concentrations were measured at 0 and 72 hours in all flasks. In addition, test item concentrations were measured in the pre-exposed flasks prior to exposure to light.

DEFINITIVE TEST
The definitive test was conducted based on the results of the range-finding test and the stability test. Tetramethylthiuram monosulfide was readily degraded by light and its degradation products were shown to be as toxic as the parent compound. Therefore, additional flasks pre-exposed to light were also inoculated and incubated under the same conditions as the "fresh flask": the first test item stock solutions (at 20 mg/L nominal concentration) was prepared. A known mass of test item (20.0 mg) was poured into a volumetric flask. The flask was then filled up to 1000 mL QSP with the test medium. The solution was kept under high speed stirring at ambient temperature during approximately 30 min. with a magnetic stir bar. Then it was incubated in the phytoculture cabinet under test conditions (temperature and light intensity) during around 68 hours (i.e. until start of the test) to generate the degradation products.

At the beginning of the test, the second test item stock solution was similarly prepared for the "fresh flasks". Algae were exposed to a series of test item concentrations agreed with the Sponsor (0.032, 0.1, 0.32, 1 and 3.2 mg/L of nominal concentrations) and prepared from successive dilutions of both fresh and old stock solutions. Same nominal concentrations were tested for both fresh and aged solutions. An inoculated control flask (labelled T) was prepared and incubated under the same conditions, with no test item. Three vessels were prepared at each test concentration and six vessels for the control group; each vessel was inoculated with 10 000 cells/mL.

Test item concentrations in the pre-exposed flasks were the same as in the series of non-pre-exposed flasks (fresh flasks) and performed in triplicate as well. During this range-finding test, test item concentrations were measured at 0 and 72 hours in all flasks. In addition, test item concentrations were measured in the pre-exposed flasks prior to exposure to light.

ENVIRONMENTAL CONDITIONS
The incubation was conducted in two phytoculture cabinets that allow test flasks to be incubated under precise conditions: the cultures were maintained at a temperature in the range of 21 to 24°C, controlled at ± 2°C; flasks were continuously shaken with a rotation at 20 rpm and constantly illuminated by fluorescent tubes between 6,000 and 10,000 lux in one cabinet or kept in the dark in the other cabinet, as appropriate.
Test vessels were made of glass bottles (around 120 mL capacity) tightly closed with bungs and without headspace. The control treatment was maintained under the same conditions, with no test item. No auxiliary substances were used.

OBSERVATIONS
Algal cell concentrations were measured in each flask at 24, 48 and 72 h using flow cytometry (Guava easyCyte™ flow cytometer Merck Millipore). Cell concentrations were determined using 96 wells single use microplates and a laser beam at 488 nm. The cytometer is calibrated every week using the Guava check kit. Before each measurement, settings were adjusted. Non-algal particles were excluded from the analysis by setting an acquisition value threshold. This threshold was set up after analysis of cytograms with no threshold where a clear discrimination was observed between the algal population and the other events. Therefore, only the events with the same size than alga were used to assess the toxic effects.

The number of events counted was set up to 1500 to 3000 depending on the algal population. To minimize the number of coincident events, the analysed cell concentration was less than 500 cells/µL. Data processing was carried out using “Insight Software” (Guava easyCyte™ flow cytometer Merck Millipore).

The pH and dissolved oxygen concentrations were measured in inoculated flasks at the beginning and the end of the test. The temperature in the incubator was continuously recorded throughout the test.
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
growth rate inhibition
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.14 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
growth rate inhibition
Details on results:
STABILITY TEST
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, slightly yellow, no precipitation was observed at the end of the test. Chemical analysis of test samples kept under darkness for 72 hours indicated that concentrations of the test item slightly decreased over the 72h test period (less than 15% decrease), while disappearance rate of test item in test samples kept under constant light was high (> 98%). Photolysis was thus confirmed. At 1mg/L, disappearance of the test item in age solution was 90%.

RANGE FINDER
The appearance of the test solutions was visually checked at the beginning and at the end of the test period. Solutions were found to be clear, slightly yellow, no precipitation was observed at the end of the test. Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm.
The analytical results showed that, as expected, aged solution did not contain any test item at 0h. Toxicity to algae was observed in the aged solutions, supporting the hypothesis according to which the test item degrades into a degradation product. EC50 for fresh and aged solutions were very similar (close to 1 mg/L). Therefore, it was concluded that test item and its degradation product toxicities were equivalent. It was therefore justified to derive EC10 and EC50 on the basis of the initial amount of test item introduced in the flask of the fresh solution group (i.e., nominal concentrations).

DEFINITIVE TEST
The appearance of the test solutions was visually checked at the beginning and at the end of the test period. Solutions were found to be clear, slightly yellow, no precipitation was observed at the end of the test. Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm.
The analytical results showed that, as expected, aged solution did not contain any test item at 0h. Toxicity to algae was observed in the aged solutions, supporting the hypothesis according to which the test item degrades into a degradation product. EC50 for fresh and aged solutions were very similar (close to 1 mg/L for aged solution and 0.3 mg/L for the test item). Therefore, it was concluded that test item and its degradation product toxicities were equivalent. It was therefore justified to derive EC10 and EC50 on the basis of the initial amount of test item introduced in the flask of the fresh solution group (i.e., nominal concentrations).
Results with reference substance (positive control):
- Results with reference substance valid? YES
- EC50: 1.57 mg/L (growth rate inhibition)
Reported statistics and error estimates:
Data were analysed with Toxrat 3.2.1 (standard template OECD 201).

Test solution

Replicate

Algal density at 24h (Fd, ¢ *104/ ml )

Algal density at 48h (Fd, ¢ *104/ ml )

Algal density at 72h (Fd, ¢ *104/ ml )

Average specific growth rate J0-J3

Specific growth rate inhibition (%)

Replicate mean

Mean

RSD

Replicate mean

Mean

RSD

Replicate mean

Mean

RSD

Replicate mean

Average

Sx

CV

Control

a

4.18

4.12

13.42

19.22

20.24

4.70

69.10

76.73

10.51

1.412

1.445

0.033

2.293

Inhibition
(per replicate)

Inhibition
(mean)

b

3.18

21.23

77.52

1.450

c

4.62

19.74

74.43

1.437

d

4.67

21.01

73.97

1.435

e

3.83

19.21

73.12

1.431

f

4.22

21.01

92.24

1.508

0.03

a

5.82

4.56

24.09

18.79

17.48

11.77

73.14

68.71

8.51

1.431

1.409

0.029

2.060

1.0

2.5

b

3.93

18.53

70.91

1.420

1.7

c

3.91

15.11

62.08

1.376

4.8

0.10

a

3.44

3.95

15.97

17.05

16.34

5.16

55.38

48.13

13.34

1.338

1.289

0.043

3.363

7.4

10.8

b

3.75

15.41

43.15

1.255

13.2

c

4.65

16.56

45.86

1.275

11.8

0.32

a

4.00

4.02

10.44

7.30

7.66

10.65

7.43

8.83

13.99

0.669

0.724

0.049

6.708

53.7

49.9

b

4.45

8.60

9.76

0.760

47.4

c

3.61

7.09

9.30

0.743

48.6

1.00

a*

 -

1.52

2.17

 -

1.58

6.21

 -

0.85

6.21

-0.054

0.021

38.696

-

103.7

b

1.54

1.51

0.89

-0.039

102.7

c

1.49

1.65

0.82

-0.068

104.7

3.20

a

1.26

1.30

3.73

0.90

0.91

5.27

0.08

0.08

4.48

-0.846

-0.835

0.015

-1.772

158.5

157.8

b

1.29

0.96

0.09

-0.818

156.6

c

1.35

0.87

0.08

-0.840

158.1

Table 1: Algal cell density and specific growth rate inhibition in Pseudokirchneriella subcapitata exposed to TMTM.

Test solution

Replicate

Algal density at 24h (Fd, ¢ *104/ ml )

Algal density at 48h (Fd, ¢ *104/ ml )

Algal density at 72h (Fd, ¢ *104/ ml )

Average specific growth rate J0-J3

Specific growth rate inhibition (%)

Replicate mean

Mean

RSD

Replicate mean

Mean

RSD

Replicate mean

Mean

RSD

Replicate mean

Average

Sx

CV

Control

a

4.18

4.12

13.42

19.22

20.24

4.70

69.10

76.73

10.51

1.412

1.445

0.033

2.293

Inhibition
(per replicate)

Inhibition
(mean)

b

3.18

21.23

77.52

1.450

c

4.62

19.74

74.43

1.437

d

4.67

21.01

73.97

1.435

e

3.83

19.21

73.12

1.431

f

4.22

21.01

92.24

1.508

0.03

a

3.37

3.19

8.05

19.73

17.31

12.26

78.66

75.85

4.05

1.455

1.443

0.014

0.941

-0.7

0.2

b

3.31

16.42

76.32

1.445

0.0

c

2.90

15.77

72.57

1.428

1.2

0.10

a

3.85

3.19

24.04

17.16

16.18

5.45

70.69

72.50

2.16

1.419

1.428

0.007

0.507

1.8

1.2

b

2.35

15.93

73.39

1.432

0.9

c

3.38

15.45

73.41

1.432

0.9

0.32

a

3.47

3.52

2.14

12.73

11.46

14.45

52.44

50.97

2.79

1.320

1.310

0.009

0.709

8.7

9.3

b

3.61

12.07

50.86

1.310

9.4

c

3.49

9.59

49.60

1.301

10.0

1.00

a

2.38

2.53

8.92

5.85

6.14

4.43

9.83

10.14

5.78

0.762

0.772

0.019

2.457

47.3

46.6

b

2.42

6.16

10.82

0.794

45.1

c

2.79

6.39

9.78

0.760

47.4

3.20

a

1.02

1.04

2.55

0.92

0.92

0.35

0.31

0.28

13.05

-0.388

-0.431

0.044

-10.153

126.9

129.8

b

1.07

0.92

0.28

-0.429

129.7

c

1.03

0.91

0.24

-0.476

132.9

Table 2: Algal cell density and specific growth rate inhibition in Pseudokirchneriella subcapitata exposed to degradation products of TMTM. Concentrations indicated in the first column reflect initial concentrations of TMTM before aging for 3 days. At the end of this period, no TMTM was detectable. However, its degradation product(s) (not monitored) clearly showed an equivalent toxicity.

Validity criteria fulfilled:
yes
Conclusions:
The test item is photodegradable. The degradation products are as toxic for algae as the parent item. EC50 and EC10 were 0.32 and 0.14 mg/L respectively according to growth rate inhibition.
Executive summary:

This study was designed to determine the effects of tetramethylthiuram monosulfide (CAS 97-74-5) (further referred as “the test item”) on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline. The test item was proved to be degraded by photolysis through pre-experiments. The definitive test was thus conducted with "fresh" and "aged solutions" in order to assess the test item toxicity as well as the toxicity of its degradation products. Aged solutions were solutions containing the test item which was let to degrade to light for a sufficient amount of time to form 100% degradation product (i.e. complete disappearance of the test item in the solutions). As the volatility of these degradation products was unknown and no analytical method was developed to identify them, all experiments were carried out in glass bottles tightly closed with bungs and without headspace to prevent evaporation.

Chemical analysis of test samples from "fresh solutions" taken at 0h confirmed the test item nominal concentrations. Chemical analysis of test samples from "aged solutions" taken at 0h confirmed that the test item was quite completely degraded by photolysis, and thus that the toxicity observed in the “aged solutions” should be attributed to the test item degradation products.The algae test results demonstrated that regardless of the tested product (test item itself or its degradation products), algal growth was inhibited at similar levels of nominal test item concentration: this indicates that test item and its degradation products have similar toxicities on algal growth. Consequently, the exposure concentrations were based on the nominal concentrations of test item. EC50 and EC10 values for growth rate inhibition were 0.32 and 0.14 mg/L.

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
0.32 mg/L
EC10 or NOEC for freshwater algae:
0.14 mg/L

Additional information

This study (Chastenet, 2016) was designed to determine the effects of tetramethylthiuram monosulfide (CAS 97-74-5) (further referred as “the test item”) on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline. The test item was proved to be degraded by photolysis through pre-experiments. The definitive test was thus conducted with "fresh" and "aged solutions" in order to assess the test item toxicity as well as the toxicity of its degradation products. Aged solutions were solutions containing the test item which was let to degrade to light for a sufficient amount of time to form 100% degradation product (i.e. complete disappearance of the test item in the solutions). As the volatility of these degradation products was unknown and no analytical method was developed to identify them, all experiments were carried out in glass bottles tightly closed with bungs and without headspace to prevent evaporation. Chemical analysis of test samples from "fresh solutions" taken at 0h confirmed the test item nominal concentrations. Chemical analysis of test samples from "aged solutions" taken at 0h confirmed that the test item was quite completely degraded by photolysis, and thus that the toxicity observed in the “aged solutions” should be attributed to the test item degradation products. The algae test results demonstrated that regardless of the tested product (test item itself or its degradation products), algal growth was inhibited at similar levels of nominal test item concentration: this indicates that test item and its degradation products have similar toxicities on algal growth. Consequently, the exposure concentrations were based on the nominal concentrations of test item. EC50 and EC10 values for growth rate inhibition were 0.32 and 0.14 mg/L.

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