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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:
2017-01-20 - 2017-03-30 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Version dated 23-March-2006
Deviations:
yes
Remarks:
Deviations to the Study Plan (see section "Any other information on materials and methods incl. tables").
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 10, 20, 40, 80, 160 mg/L
- Sampling method: Specimens were drawn from freshly prepared treatments and those incubated for 72 hr.
- Sample storage conditions before analysis: The supporting analysis of the t0 specimens could not be performed on the same day of collection of the specimens. Therefore, these specimens from freshly prepared test solutions were stored at 2-8°C and then analysed. t72hr specimens were analysed on the day of collection from the treatments.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was introduced into the test solutions as follows: First, solutions of the test item were individually prepared for each test concentration in ultrapure water and stirred on a magnetic stirrer for 10 min. at room temperature. Thereafter the solutions were transferred into 10 test vessels, respectively. By the addition of the "intermediate dilution" and the algal inoculation (or diluted „intermediate dilution“), the final nominal concentrations (see below) were achieved with 50 mL of final volume.

For the inoculated test solutions with the test item, as well as those without any test item, seven replicates for the main test and five replicates for the screening tests were incubated under constant light. As a reagents’ Blank without algal inoculation, three test solutions were prepared as described above containing a 1:10 diluted intermediate dilution instead of the algal pre-culture. These blanks were incubated under the same conditions under constant light as the test solution to be tested. The measured turbidity in those test solutions was subtracted from those with the algal inoculation. In the same way, seven control solutions without any test item but with algal inoculation were prepared within the main test as well as one test solution without any test item and without algal inoculation. For better comprehension, the exact composition of the test solutions is given in the table below

For the screening test, the following suspensions with the test item were prepared as described above:
[1] 1.26 mg / 1000 mL ultrapure water (resulting 1 mg/L final nominal conc.)
[2] 12.5 mg / 1000 mL ultrapure water (resulting 10 mg/L final nominal conc.)
[3] 125.0 mg / 1000 mL ultrapure water (resulting 100 mg/L final nominal conc.)

The treatment of the stock solutions was made as indicated above. A table including the composition of treatments for the screening test is presented in section "Any other information on materials and methods incl. tables".

For the definitive main test , the following amounts of suspensions with the test item were prepared as described above:
[1] 25.0 mg / 2000 mL ultrapure water (resulting 10 mg/L final nominal conc.)
[2] 50.0 mg / 2000 mL ultrapure water (resulting 20 mg/L final nominal conc.)
[3] 100.0 mg / 2000 mL ultrapure water (resulting 40 mg/L final nominal conc.)
[4] 200.0 mg / 2000 mL ultrapure water (resulting 80 mg/L final nominal conc.)
[5] 400.0 mg / 2000 mL ultrapure water (resulting 160 mg/L final nominal conc.)

The treatment of the stock solutions was made as indicated above. A table including the composition of treatments for the definitive test is presented in section "Any other information on materials and methods incl. tables".

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen [SAG], Strain-No. 86.81
- Age of inoculum (at test initiation): Three days prior to starting the test, a pre-culture was inoculated using the algal stock culture.
- Method of cultivation: The algae were cultivated under aseptic / sterile conditions.

ACCLIMATION
- Acclimation period: Three days before starting of the test (main test), an algal pre-culture was made which was inoculated from an algal stock culture derived from the algal type culture collection Göttingen in order to guarantee sterile conditions at all.
- Culturing media and conditions (same as test or not): The pre-culture was incubated under the same conditions as the test solutions to be tested afterwards.
- Any deformed or abnormal cells observed: none stated
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
25.8 - 26.8 °C (measured with a data logger [Elpro Ecolog])
pH:
Control with algal inoculation: 8.30 - 9.74
Control without algal inoculation: 8.28 - 8.42
10 mg/L with algal inoculation: 7.80 - 9.79
10 mg/L without algal inoculation: 7.78 - 8.35
20 mg/L with algal inoculation: 7.51 - 9.54
20 mg/L without algal inoculation: 7.48 - 8.30
40 mg/L with algal inoculation: 7.17 - 9.83
40 mg/L without algal inoculation: 7.14 - 8.24
80 mg/L with algal inoculation: 6.78 - 8.14
80 mg/L without algal inoculation: 6.70 - 8.03
160 mg/L with algal inoculation: 5.94 - 7.23
160 mg/L without algal inoculation: 5.90 - 7.33
Nominal and measured concentrations:
Nominal: 0, 10, 20, 40, 80, 160 mg/L
Found (t0): 0, 10.4, 20.9, 40.8, 83.6, 166.6 mg/L
Found (t72h): 0, 10.4, 20.7, 41.4, 83.1, 166.3 mg/L
Mean measured concentration (0-72h): 0, 10.4, 20.8, 41.1, 83.3, 166.4 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL-glass cylinders
- Material, size, headspace, fill volume: heat-sterilized glass, 50 mL final volume
- Initial cells density: 3*10E3
- Control end cells density: 5.9*10E5
- No. of vessels per concentration (replicates): 10 (main test) / 8 (screening)
- No. of vessels per control (replicates): 10 (main test) / 8 (screening)

GROWTH MEDIUM
- Standard medium used: yes, but the concentration of NaHC03 was increased to be twice of that indicated in the Guideline 201, as this concentration has been found to be optimal.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The mineral nutrient medium was prepared from the following three stock solutions and NaHCO3:

A) Mineral Nutrient Salts:
1.5 g NH4Cl A0165045227 Merck
1.2 g MgCl2 x 6 H2O A799733043 Merck
1.8 g CaCl2 x 2 H2O A0744882504 Merck
1.5 g MgSO4 x 7 H2O A0457586240 Merck
0.16 g KH2PO4 A0761277513 Merck
were dissolved in 1 L deionised water (abbreviated “DI-H2O” hereafter) and autoclaved

B) FeCl3-Solution:
0.064 g FeCl3 x 6 H2O 15D270031 VWR
0.10 g Na2EDTA x 2 H2O K45714718432 Merck
was dissolved in 1 L DI-H2O and used freshly on the day of preparation.

C) Trace Elements:
185 mg H3BO3 A0724165431 Merck
415 mg MnCl2 x 4 H2O A0536427427 Merck
3 mg* ZnCl2 B0974516344 Merck
1.5 mg* CoCl2 x 6 H2O B0744239219 Merck
0.01 mg* CuCl2 x 2 H2O K45082633421 Merck
7 mg* Na2MoO4 x 2 H2O A0751921520 Merck
were dissolved in 1 L DI-H2O and autoclaved. To include components marked "*", solutions were prepared by an additional dilution step using 1 mL of 300 mg ZnCl2/100 mL DI-H2O, 1 mL of 150 mg CoCl2*6 H2O/100 mL DI-H2O, 100 µL of 10 mg CuCl2 * 2 H2O/100 mL DI-H2O, and 2 mL of 350 mg Na2MoO4 * 2 H2O/100 mL DI-H2O.

D) Sodium Hydrogen Carbonat:
1.00 g/L NaHCO3 K 44832529410 Merck
was introduced in the solid form directly into the "intermediate dilution" which then was sterilized by ultrafiltration. The final concentration of NaHCO3 was twice as concentrated as indicated in the OECD Guideline 201.

Ultrapure water generating: Purelab Classic D1, USF Seral; Ser.-No. 1470984-10 01 (ELGA Labwater)

- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous
- Light intensity and quality: illumination rate of > 120 uE/m2s ([=<8000 Lux]

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer, daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 (main test), 10 (screening test)
- Range finding study: 0, 1, 10, 100 mg/L
- Test concentrations: 0, 10, 20, 40, 80, 160 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
55.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: Results should be based on the nominal concentrations applied
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
27.37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: Results should be based on the nominal concentrations applied
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18.99 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: Results should be based on the nominal concentrations applied
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Results should be based on the nominal concentrations applied
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
82.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Results should be based on the nominal concentrations applied
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
32.86 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Results should be based on the nominal concentrations applied
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
40 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: both growth rate and yield
Remarks on result:
other: Results should be based on the nominal concentrations applied
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
both growth rate and yield
Remarks on result:
other:
Remarks:
Results should be based on the nominal concentrations applied
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none stated
- Unusual cell shape: none stated
- Colour differences: none stated
- Flocculation: not stated
- Aggregation of algal cells: not stated
- Any stimulation of growth found in any treatment: For the parameter yield an activation of 8.2 and 5.7% was determined at the two lowest main test concentrations of 10 and 20 mg/L compared to the control, respectively. For the parameter growth rate an activation of 1.5 and 1.0% was determined at the two lowest main test concentrations of 10 and 20 mg/L compared to the control, respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not required
Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to OECD TG 201 with no relevant deviations. The results were so obtained via a scientifically reasonable method. The validity criteria for the study as given by the OECD Guideline 201 were all met and the study is therefore considered to be valid. Hence, there is no doubt that the obtained results are not reliable: The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The test was performed using five concentrations in the range of 10 to 160 mg/L.
As a conclusion of the analytical part of this study, it can be stated that the concentrations of 2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) remained sufficiently stable during incubation of 72 hr: after 72 hr of incubation recoveries of 99.0 to 101.5 % of the initial values measured at time t0 were obtained in the treatments after 72 hr. Based on this, the reported effect concentrations (EC values) should refer to nominal concentrations of the test item.

The following EC-values (72h; growth rate; nominal concentrations) were determined accordingly:
LOEC 40 mg/L
NOEC 20 mg/L
EC50 > 160 mg/L
Based on these results, the test item does not need to be classified as acute or chronic toxic to the aquatic environment.
Executive summary:

The toxicity of 2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) towards algae was tested according to OECD-Guideline No. 201, in the Version dated 23-March-2006, under GLP. The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The test was performed using five concentrations in the range of 10 to 160 mg/L. The following EC-values were obtained:

On Basis of the Nominal Concentrations

[mg test item/ L]

Yield (0 - 72hr)                                

                           EC10          18.99

          95%-CL    lower          18.14

                           upper          19.87 

                           EC20          27.37

           95%-CL    lower          26.17

                           upper          28.65 

                           EC50          55.11

           95%-CL    lower          52.04

                           upper          58.25 

                 Yield  LOEC          40.00

                          NOEC          20.00

Section-by-section growth rate (0-72hr)

                           EC10         n.d. (>160)

           95%-CL    lower         n.d. (>160)

                           upper         n.d. (>160)

                           EC20         n.d. (>160)

          95%-CL    lower         n.d. (>160)

                           upper         n.d. (>160)

Section-by-section growth rate (0-72hr)

                            EC50          n.d. (>160)

           95%-CL    lower           n.d. (>160)

                           upper           n.d. (>160)

                          LOEC           n.d. (>160)

                          NOEC           n.d. (>160)

Growth rate (0 - 72 hr)                    

                           EC10          32.86

           95%-CL    lower          16.65

                           upper          64.85 

                           EC20          82.35

           95%-CL    lower          39.56

                           upper        175.72 

                           EC50          n.d. (>160)

           95%-CL    lower           n.d. (>160)

                          upper           n.d. (>160)

     Growth rate  LOEC          40.00

                          NOEC          20.00

n.d.: not determined due to mathematical reasons or inappropriate data

As a conclusion of the analytical part of this study, it can be stated that the concentrations of 2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) remained sufficiently stable during incubation of 72hr:after 72 hr of incubation recoveries of 99.0 to 101.5 % of the initial values measured at time t0 were obtained in the treatments after 72 hr. Based on this, the reported effect concentrations (EC values) should refer to nominal concentrations of the test item.

The validity criteria for the study as given by the OECD Guideline 201 were all met and the study is therefore considered to be valid.

Description of key information

Toxicity to algae: NOEC (72h, growth rate & yield) = 20 mg/L, LOEC (72h, growth rate & yield) = 40 mg/L, EC10 (yield) = 18.99 mg/L, EC10 (growth rate) = 32.86 mg/L, EC50 (yield) = 55.11 mg/L and EC50 (growth rate) > 160 mg/L for Desmodesmus subspicatus (OECD 201, EU method C.3, static)

Key value for chemical safety assessment

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

Additional information

There is a Klimisch 1 (OECD 201 on Desmodesmus subspicatus over 72h) available. In this study, the highest concentration tested was 160 mg/L, and the results are: NOEC (72h, growth rate & yield) = 20 mg/L, LOEC (72h, growth rate & yield) = 40 mg/L, EC10 (yield) = 18.99 mg/L, EC10 (growth rate) = 32.86 mg/L, EC50 (yield) = 55.11 mg/L and EC50 (growth rate) > 160 mg/L. Based on this data, there is no classification as hazardous to the aquatic environment, neither acute nor chronic, according to Regulation (EC) No 1272/2008 and amendments, triggered. There is no indication given that this data is not relevant for the assessment of ecotoxicological properties of DMTD. Hence, no data gap was identified, and the key values, EC50 (growth rate) > 160 mg/L and NOEC (72h, growth rate & yield) = 20 mg/L, can be chosen for further risk assessment.