(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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 (CAS 51988-14-8) in unbuffered water at room temperature, hence posing the same conditions as tests 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. Based on this, it can be concluded that the registered 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 the hydrolysis products 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0). According to the “Read-Across Assessment Framework” (RAAF) (ECHA 2017) the analogue approach is followed. The basis for this analogue approach is the “(Bio)transformation to common compound(s)” (scenario 1). “This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst-case approach” (ECHA 2017). The target substance 5-Mercapto-1,3,4-thiadiazole-2-thiol-benzoate (CAS 51988-14-8) is rapidly hydrolysed to 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0). Therefore, the proposed source substances for the target substance 5-Mercapto-1,3,4-thiadiazole-2-thiol-benzoate are its final hydrolysis products DMTD and benzoic acid.

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. There are no non-common compounds, which have to be taken into account in this read-across approach.
The performed approach is further supported by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (Series on Testing and Assessment No. 23, 8 Feb 2019 - ENV/JM/MONO(2000)6/REV1). In this document under point 82 it is stated that “If the test chemical is likely to be unstable, a decision to test the parent test chemical and/or its degradation products, if identified, should be based on a consideration of its half-life under test and real-world conditions. The following decision criteria are suggested only as a guide for static and static-renewal exposure systems with test solution renewal intervals of 24 hours:
• Half-life >3 days: test parent chemical;
• Half-life < 3 days and > 1 hour: consider on a case-by-case basis, and include possible testing of degradation products;
• Half-life < 1 h: test degradation products.”

Having a half-life of 1.91 h the registered substance belongs to the category of ‘< 3 days and > 1 hour’. Thus, the testing strategy has to be decided on a case-by-case basis. With 1.91 h 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. Also, the value is close to the threshold of 1 h. Therefore, the registrant concluded that the substance already qualifies to hydrolyze rapidly, and testing of hydrolysis products is suitable.
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 2 h) 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 DMTD, so this substance can be regarded as worst-case for the target substance and it 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 registered 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 for cross-reference:

read-across source

Post exposure observation period:

none

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

4.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3.83 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: no immobilisation noted
- Other adverse effects control: none stated
- 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):

- Results with reference substance valid? yes
- Limit test: no
- Dose-response test: yes
- ECx: EC50 (24 hr) = 0.95 mg/L
- Other: In order to verify the sensitivity of the test system towards toxic items the effect of potassium dichromate towards different animals of this breed was tested prior to beginning of the test. The value of the EC50 (24 hr) in this period of time was at 0.95 mg/L potassium dichromate. This value is in accordance with the recommendations of 0.6 to 2.1 mg/L K2Cr2O7 given in OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test).

Reported statistics and error estimates:

Statistical evaluation was done using the commercial computer program ToxRat Professional Version 3.2.1.

Validity criteria fulfilled:

yes

Conclusions:

The study was performed according to OECD TG 202 without deviations which may have impact on the validity of the study. Thus, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable:
Under the conditions used for the test, there was a significant immobilisation of the daphnias at concentrations >2.0 mg/L (definitive main test).
As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following EC-values were determined accordingly after 48 hours:
Mobility:
LOEC = 4.0 mg/L
NOEC = 2.0 mg/L
EC50 = 5.95 mg/L
Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the facts that the test item is soluble in water, i.e. above the determined EC50 value, and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 2. As 5-Mercapto-1,3,4-thiadiazol-2-thiol-benzoate hydrolyzes into a mixture of hydrolysis products, which contains DMTD > 25%, the registered substance as such should be classified as aquatic chronic Cat. 2, applying the rules for classification as mixtures under Regulation 12072/2008.

Executive summary:

2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) was tested for acute toxicity towards daphnia according to OECD-Test Guideline 202 (version dated 13-Apr-2004) in compliance with GLP. In order to investigate the influence of the test item towards the daphnia, the swimming behaviour of the animals was recorded. Under the conditions used for the test, there was an immobilisation of the daphnia at concentrations >2 mg/L. As a conclusion of the analytical part of this study, it can be stated that the concentrations 2,5-Dimercapto-1,3,4-thiadiazole (“DMTD”) remained sufficiently stable during incubation of 48hr. As a result of the supporting analysis it can be stated that the results of the biological part should be based on the nominal concentrations applied because the recoveries of the nominal concentrations were all >80%.

 

The following EC-values (effective concentrations) were determined accordingly:

	Basis: Nominal Concentrations [mg/L]
	t24hr	t48hr
NOEC	≥ 10.00	2.00
LOEC	>10.00	4.00
EC10	7.57	3.83
EC20	11.71	4.46
EC50	n.d. (>10.00)	5.95

n.d. = not determined for mathematical reasons.

 

The test is considered valid as all conditions for validity were met.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Literature data

Adequacy of study:

weight of evidence

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 (CAS 51988-14-8) in unbuffered water at room temperature, hence posing the same conditions as tests 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. Based on this, it can be concluded that the registered 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 the hydrolysis products 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0). According to the “Read-Across Assessment Framework” (RAAF) (ECHA 2017) the analogue approach is followed. The basis for this analogue approach is the “(Bio)transformation to common compound(s)” (scenario 1). “This scenario covers the analogue approach for which the read-across hypothesis is based on (bio) transformation to common compound(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst-case approach” (ECHA 2017). The target substance 5-Mercapto-1,3,4-thiadiazole-2-thiol-benzoate (CAS 51988-14-8) is rapidly hydrolysed to 1,3,4-Dimercapto-thiadiazol (DMTD, CAS 1072-71-5) and benzoic acid (CAS 65-85-0). Therefore, the proposed source substances for the target substance 5-Mercapto-1,3,4-thiadiazole-2-thiol-benzoate are its final hydrolysis products DMTD and benzoic acid.

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. There are no non-common compounds, which have to be taken into account in this read-across approach.
The performed approach is further supported by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (Series on Testing and Assessment No. 23, 8 Feb 2019 - ENV/JM/MONO(2000)6/REV1). In this document under point 82 it is stated that “If the test chemical is likely to be unstable, a decision to test the parent test chemical and/or its degradation products, if identified, should be based on a consideration of its half-life under test and real-world conditions. The following decision criteria are suggested only as a guide for static and static-renewal exposure systems with test solution renewal intervals of 24 hours:
• Half-life >3 days: test parent chemical;
• Half-life < 3 days and > 1 hour: consider on a case-by-case basis, and include possible testing of degradation products;
• Half-life < 1 h: test degradation products.”

Having a half-life of 1.91 h the registered substance belongs to the category of ‘< 3 days and > 1 hour’. Thus, the testing strategy has to be decided on a case-by-case basis. With 1.91 h 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. Also, the value is close to the threshold of 1 h. Therefore, the registrant concluded that the substance already qualifies to hydrolyze rapidly, and testing of hydrolysis products is suitable.
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 2 h) 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 DMTD, so this substance can be regarded as worst-case for the target substance and it 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 registered 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 for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.04 mmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% C.I. = 6.25–7.85

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

860 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Validity criteria fulfilled:

not specified

Conclusions:

Testing was sufficiently documented and performed similar to OECD 202. Thus, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable: Under the conditions used for the test, the EC50 (48h) of benzoic acid was determined to be 7.04 (6.25–7.85) mmol/l, i.e. 860 mg/l.
Based on these results, benzoic acid does not need to be classified as toxic to the aquatic environment, neither acute nor chronic, according to Regulation 1272/2008. The same applies for the target substance 5-Mercapto-1,3,4-thiadiazol-2-thiol-benzoate.

Executive summary:

The 48 –hr-acute toxicity of benzoic acid to daphnia magna was studied under static conditions.  Daphnids were exposed to the test chemical at nominal concentrations of 1.25–20 mmol/l for 48 hr.  Immobilization was observed daily.  The 48– hour EC₅₀ was 7.04 (6.25–7.85) mmol/l, i.e. 860 mg/l.

Based on the results of this study, benzoic acid does not need to be classified as toxic to daphnids in accordance with the classification system of Regulation 1272/2008.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Description of key information

Read across to the Benzoic acid: OECD 202, Daphnia magna, static, EC50(48h): 7.04 (6.25–7.85) mmol/L, i.e. 860 mg/L.

Read across to DMTD: OECD 202, GLP compliance, Daphnia magna, static, EC50(48h): 5.95 mg/L, NOEC = 2.0 mg/L

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

5.95 mg/L

Additional information

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).

Short term toxicity to aquatic invertebrates for DMTD

The study was performed according to OECD TG 202 without deviations which may have impact on the validity of the study. Thus, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable:
Under the conditions used for the test, there was a significant immobilisation of the daphnias at concentrations >2.0 mg/L (definitive main test).
As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following EC-values were determined accordingly after 48 hours:
Mobility:
LOEC = 4.0 mg/L
NOEC = 2.0 mg/L
EC50 = 5.95 mg/L

Short-term toxicity to aquatic invertebrates for Benzoic acid

The 48 hr acute toxicity of benzoic acid to daphnia magna was studied under static conditions.  Daphnids were exposed to the test chemical at nominal concentrations of 1.25–20 mmol/L for 48 hr.  Immobilization was observed daily.  The 48hour EC50 was 7.04 (6.25–7.85) mmol/L, i.e. 860 mg/L.