1,2,4-triazole-3-thiol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

The following deviations were observed:
• The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.
• The temperature in the experiment was in the range 23.0 – 25.5 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and expo-nential growth was observed in the blank control this was stated as uncritical.
• The temperature during pre-culture incubation was in the range 22.8 – 24.7 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncriti-cal.
• The pre-culture was prepared 5 days before the test start. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

Deviations:

yes

Remarks:

see Version/Remarks

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-triazole-3-thiol

EC Number:

221-656-6

EC Name:

1,2,4-triazole-3-thiol

Cas Number:

3179-31-5

Molecular formula:

C2H3N3S

IUPAC Name:

4H-1,2,4-triazole-3-thiol

Test material form:

solid: crystalline

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution containing 100.6 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution of this stock solution with algal medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

-

Test temperature:

23.0 – 25.5 °C

pH:

6.7-8.8

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

The measured concentrations lay between 104 % and 106 % of the nominal concentrations at the beginning of the test and between 106 % and 118 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the nominal concentrations (see § 176 Guidance Doc. No. 23).

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

62.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Any other information on results incl. tables

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. The details of calculation are stated in chapter 19.

1.1     Biological Results

The following values were determined:

Table10.1‑a    Biological Results of the Test Item

Parameter	Value	95 % confidence interval
NOEC (Growth Rate) 72 h	< 3.2 mg/L	--
NOEC (Yield) 72 h	< 3.2 mg/L	--
LOEC (Growth Rate) 72 h	≤3.2 mg/L	--
LOEC (Yield) 72 h	≤3.2 mg/L	--
72h ErC10	11.93 mg/L	9.36 – 15.19 mg/L
72h EyC10	1.44 mg/L	0.69 – 3.03 mg/L
72h ErC50	62.42 mg/L	46.49 – 82.94 mg/L
72h EyC50	10.64 mg/L	4.60 – 24.39 mg/L

 

 

 

 

2      Validity

2.1     Increase Factor

The cell concentration in the blank control should increase by a factor of at least 16 within 72h.

2.2     Daily Growth Rates

Mean coefficient of variation of daily growth rates in the blank controls should be 35 % at the most.

Coefficient of variation of average growth rate during the whole test period should be 7 % at the most.

2.3     Values and assessment

The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined. Values and assessment can be found in the following tables.

Table11.3‑a    Daily Growth Rates of the blank controls

Growth rates	days 0 – 1	days 1 – 2	days 2 – 3	CV of sectional daily growth rates	days 0 – 3
Replicate 1	1.633	1.715	1.825	6%	1.724
Replicate 2	1.928	1.586	1.781	10%	1.765
Replicate 3	1.670	1.764	1.840	5%	1.758
Replicate 4	1.769	1.612	1.832	7%	1.738
Replicate 5	1.527	2.053	1.644	16%	1.741
Replicate 6	1.614	1.864	1.733	7%	1.737
Mean	1.690	1.766	1.776	8%	1.744
Standard deviation	0.140	0.174	0.076		0.015
CV	8%	10%	4%		1%

CV = Coefficient of Variation

 

Table11.3‑b    Assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	187	Valid
Mean coefficient of variation of daily growth rates	max. 35 %	8%	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7 %	1%	Valid

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed. The study was performed using 5 concentrations ranging from 1.0 to 100 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concen-tration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed at all tested concentrations. The lowest concentration showed a turbidity after 72 h incubation. Hence measurement of the cell number was biased which result in implausible high inhibition values. Turbidity in this treatment was not caused by the test item because the measured test item concentration in all treatments were in the expected range. Therefore, this treatment is excluded from the evaluation. At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The measured concentrations lay between 104 % and 106 % of the nominal concentrations at the beginning of the test and between 106 % and 118 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the nominal concentrations (see § 176 Guidance Doc. No. 23). The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory. The pH of the blank control should not fluctuate by more than 1.5 units. The change was 1.4 units in the blank control. All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Executive summary:

One valid experiment was performed. The study was performed using 5 concentrations ranging from 1.0 to 100 mg/L (nominal). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield[1] were determined from the cell number at the respective observation times. Significant inhibition of algal growth was observed at all tested concentrations. The lowest concentration showed a turbidity after 72 h incubation. Hence measurement of the cell number was biased which result in implausible high inhibition values. Turbidity in this treatment was not caused by the test item because the measured test item concentration in all treatments were in the expected range. Therefore, this treatment was excluded from the evaluation. At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The measured concentrations lay between 104 % and 106 % of the nominal concentrations at the beginning of the test and between 106 % and 118 % of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the nominal concentrations (see § 176 Guidance Doc. No. 23). The 72h-EC₅₀values of potassium dichromate (K₂Cr₂O₇, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.65 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item3-Mercapto-1,2,4-triazolewere determined:

Table 3‑a        Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	< 3.2 mg/L	≤3.2 mg/L	11.93 mg/L	62.42 mg/L
Yield	< 3.2 mg/L	≤3.2 mg/L	1.44 mg/L (extrapolated)	10.64 mg/L

Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC₁₀which is read from the graph toxicity vs. concentration may lie lower.

[1]Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period. Calculation see under 8.2 Growth and growth inhibition are quantified from measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/L test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used.