Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-01-20 - 2017-03-30 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see target record
Cross-reference
Reason / purpose:
read-across source
Remarks:
link to target
Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Klimisch 1 source record, but performed on read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
There is data available on the hydrolysis behaviour of the registered substance 5-Mercapto-1,3,4-thiadiazole-2-thiol-benzoate in unbuffered water at room temperature, hence posing the same conditions as test on aquatic toxicity. The hydrolytic degradation of 5-mercapto-1,3,4-thiadiazole-2-thiol-benzoate can be described as a pseudo-first order kinetic and the half-life of 5-mercapto-1,3,4-thiadiazole-2-thiol-benzoate was determined to be 1.91 h. 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0) were identified as degradation products as expected by hydrolytical cleavage of the thioester group. It can be concluded that the substance hydrolyzes very rapidly in water, i.e. the half life is max. 1/25 of the test duration when put into aqueous test systems, e.g. daphnia or algae acute toxicity tests. Hence, the substance as such does not need to be tested in those systems, it is sufficient for hazard assessment to use data gained with 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0).


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target chemical: Benzenecarbothioic acid, S-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) ester, CAS 51988-14-8, SMILES Code O=C(Sc(nnc1S)s1)c(cccc2)c2
Source chemical 1: Benzoic acid, CAS 65-85-0, SMILES Code O=C(O)c(cccc1)c1
Source chemical 2: 1,3,4-Thiadiazolidine-2,5-dithione, CAS 1072-71-5, SMILES Code N(NC(=S)S1)C1=S
All substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.


3. ANALOGUE APPROACH JUSTIFICATION
5-Mercapto-1,3,4-thiadiazole-2-thiol-benzoate was shown to hydrolyze rapidly in unbuffered water at room temperature, which simulates rather closely the conditions in aquatic toxicity tests. The hydrolytic degradation of 5-mercapto-1,3,4-thiadiazole-2-thiol-benzoate can be described as a pseudo-first order kinetic and the half-life of 5-mercapto-1,3,4-thiadiazole-2-thiol-benzoate was determined to be 1.91 h. 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0) were identified as degradation products as expected by hydrolytical cleavage of the thioester group. No further assessment of the hydrolysis of benzoic acid and DMTD has been conducted because these substances are known to be hydrolytically stable.

According to ECHA’s Guidance R.6, “in the context of risk assessment, (Q)SAR and read-across approaches have been used to: … estimate environmental fate data, especially partitioning behaviour and abiotic degradation (e.g. atmospheric oxidation and hydrolysis);“ Further, for data collection, as a first step, a „Preliminary analysis of reactivity, uptake and fate“ should be performed. „A preliminary assessment of expected reactivity, uptake and fate is performed on the basis of the information for the abiotic and biotic reactions involving the parent compound. The following considerations should be taken into account: …
- what chemical reactivity (what type(s) of reactions) is expected for the parent compound
- which metabolites and reaction products (i.e. hydrolysis products) are generated“ „The prelminary analysis of uptake and fate is used to determine which compound(s) (parent compound and/or reaction products and/or metabolites) are suitable for modelling the endpoint of interest.
Especially in the context of a category development for read-across, not only Metabolic pathways may serve to establish a category, but also, as a kind of sub-group, degradation processes. „On the other hand the same concept underlying the metabolic pathways can be used for environmental degradation processes. For example, for a substance which hydrolyses very rapidly in aquatic test systems (half-life <1 hour), the aquatic toxicity endpoints can be covered by the test results with the degradation product(s). However, the referenced guidance document (OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures No. 23, ENV/JM/MONO(2000)6) already lists substances as difficult for testing with a half-life < 24h („Data profile for review of test substance properties and indicator values of difficulties for media preparation and testing… Hydrolysis half-life at 25°C and pH=7 = <24 hours“).

It can be concluded that the substance hydrolyzes very rapidly in water, i.e. with 1.91h the half life is max. 1/25 of the test duration when put into aqueous test systems, e.g. daphnia or algae acute toxicity tests. It is well noted that ECHA’s guidance mentions a half-life <1 hour for the qualification as rapid in terms of hydrolysis, but as this can be considered as guidance value and the original OECD publication mentions < 24h as a boundary value for substances difficult to test, the registrant concludes that, with a half life of 1.91h, the substance already qualifies to hydrolyze rapidly.

Further, with regard to the available data on aquatic toxicity, for better comparison the estimated values are regarded first, it can be shown that the presence of the parent compound (less than 50% of the initial quantity after 2h) has no impact on the results of the aquatic toxicity tests, at least none which may underestimated the actual hazard. Besides, it can be assumed that the target substance already degrades to a certain extend during preparation of the hypothetical test solutions, even if precautions as cited in ENV/JM/MONO(2000)6 are taken. As can be seen in the section 4 Data matrix, the EC50 or LC50 values vary over a broad range. However, the lowest values were estimated for the hydrolysis product 1,3,4-Dimercapto-thiadiazol, so only this product is most relevant for a possible classification, not the parent compound. Although no actual test data on the parent compound is available, available test data on the hydrolysis products indicate that DMTD is the more toxic one, which supports the above-mentioned conclusion, as estimated data reveal the same trend in magnitudes.

Hence, the substance as such does not need to be tested in those systems, it is sufficient for hazard assessment to use data gained with 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0).


4. DATA MATRIX
The following information was estimated via US EPA EpiSuite estimation tool or taken from actual data sources:

Property CAS 51988-14-8 (target) CAS 65-85-0 (source 1) CAS 1072-71-5 (source 2)
Water solubility (EpiSuite) 395.1 mg/L 2493 mg/L 2.638e+005 mg/L
logPow (EpiSuite) 2.05 1.87 -0.6349
Biodegradability (EpiSuite) not readily biodegradable readily biodegradable not readily biodegradable
Acute toxicity fish, 96h LC50 (EpiSuite) 31.672 mg/l (Esters) 1300.781 mg/l (Neutral Organics-acid) 12.705 mg/l (Hydrazines)
8.588 mg/l (Thiols and Mercaptans) 0.624 mg/l (Thiocarbamate, Di(Substit))
187.325 mg/l (Neutral Organic SAR) 28692.277 mg/l (Neutral Organic SAR)
Acute toxicity daphnids, 48h LC50 (EpiSuite) 65.467 mg/l (Esters) 730.075mg/l (Neutral Organics-acid) 109.112 mg/l (Hydrazines)
1.073 mg/l (Thiols and Mercaptans) 1.754 mg/l (Thiocarbamate, Di(Substit))
106.884 mg/l (Neutral Organic SAR) 12773.367 mg/l (Neutral Organic SAR)
Acute toxicity algae, 96h EC50 (EpiSuite) 27.631 mg/l (Esters) 518.374 mg/l (Neutral Organics-acid) 2.622 mg/l (Hydrazines)
0.748 mg/l (Thiols and Mercaptans) 0.190 mg/l (Thiocarbamate, Di(Substit))
81.240 mg/l (Neutral Organic SAR) 3479.678 mg/l (Neutral Organic SAR)
Acute toxicity daphnids, 48h EC50 (test data) 860 mg/l 5.95 mg/L
Acute toxicity algae (test data) Inhibition starts at 1630 mg/l (96 hr) (pH = 7)
EC50 (14d) = >10 mg/l EC50 (72h) > 160 mg/L
Reason / purpose:
read-across source
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: continuous
- 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. The same applies for the target substance 5-Mercapto-1,3,4-thiadiazol-2-thiol-benzoate.
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Sampling and analysis

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.

Test solutions

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

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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

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: continuous
- 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

Results and discussion

Effect concentrationsopen allclose all
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

Applicant's summary and conclusion

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 t0were 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.