Thiophanate-methyl

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-11-11 to 2002-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

June 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L
- Sampling method: Each 100 mL OECD medium contained acetone at the rate of 0.1 mL/L was mixed well with each standard solution (1.0 mL) and the mixture was subjected to HPLC determination.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Original test solution, 1000 mg/L, was prepared by weighing 100 mg of test substance into a 100 mL volumetric flask and dissolving and diluting to the mark with acetone. Aliquots (0.156, 0.313, 0.625 and 1.25 mL) of the original test solution were transferred into 250 mL Erlenmeyer flasks respectively, and air-dried at room temperature.
One hundred milliliter OECD medium contained acetone at the rate of 0.1 mL/L was transferred into the Erlenmeyer flask and dissolved by ultrasonic bath in order to prepare the test solutions of 1.56, 3.13, 6.25 and 12.5 mg/L concentrations. As for the preparation of the test solutions of 25.0, 50.0 and 100 mg/L, aliquots (2.50, 5.00 and 10.0 mg) of the test substance were weighed into 250 mL Erlenmeyer flasks respectively, and 100 mL OECD mediums contained acetone at the rate of 0.1 mL/L was transferred into the Erlenmeyer flasks and then dispersed the media by ultrasonic bath.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: ATCC 22662
- Source: University of Boulevard (USA)

ACCLIMATION
- Acclimation period: 120 hours under the similar condition of the definitive test.
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 °C

pH:

7.8 - 9.8

Nominal and measured concentrations:

Nominal concentration: 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L
Measured Concentration: 0.832, 2.05, 4.54, 9.20,19.2, 44.0 and 90.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL
- Initial cells density: ca. 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 4,000 ~ 4,050 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx. 2 mg/L
- Range finding study: yes
- Test concentrations: 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Results based on re-calculated endpoints

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

12.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Results based on re-calculated endpoints

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.74 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Results based on re-calculated endpoints

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Results based on re-calculated endpoints

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.93 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Results based on re-calculated endpoints

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Results based on re-calculated endpoints

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 25.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Results based on algal toxicity study with the active substance (Saito, 2002, Doc. No. 823-003)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

14.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Results based on algal toxicity study with the active substance (Saito, 2002, Doc. No. 823-003)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Results based on algal toxicity study with the active substance (Saito, 2002, Doc. No. 823-003)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

9.12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Results based on algal toxicity study with the active substance (Saito, 2002, Doc. No. 823-003)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Results based on algal toxicity study with the active substance (Saito, 2002, Doc. No. 823-003)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.38 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Results based on algal toxicity study with the active substance (Saito, 2002, Doc. No. 823-003)

Details on results:

The re-evaluation of the study (necessary due to an update of the guideline requirements) with the statistic programme ToxRat® Professional (version 2.10) showed that the study results accurately reflect the sensitivity of Pseudokirchneriella subcapitata to Thiophanate-methyl under testing conditions as the concentration response was well-defined and allowed a clear definition of the EC values.

Results with reference substance (positive control):

EC50 value (95% confidence limits) of the potassium dichromate at 72 and 96 hours to the unicellular freshwater green alga Pseudokirchneriella subcapitata are 0.55 (0.50 - 0.61) and 0.47 (0.43 - 0.52) mg/L, respectively. The background data of EC50 value at 72 hours in our laboratory ranged from 0.43 to 0.56 (n = 7). The result of EC50 value at 72 hours in this study was in the region of the background data of the laboratory.

Table 1: Measured Thiophanate-methyl concentrations in the exposure solutions during the 96-hour exposure of Pseudokirchneriella subcapitata

Concentration [mg a.s./L]	Measured concentration [mg/L] *
	Dissolved test substance			Including undissolved particles
	0 hours	96 hours	Mean	0 hours	96 hours	Mean
Control	< 0.200	< 0.200	< 0.200	< 0.200	< 0.200	< 0.200
Solvent control	< 0.200	< 0.200	< 0.200	< 0.200	< 0.200	< 0.200
1.56	1.38	0.386	0.780 (50)	1.40	0.443	0.832 (53)
3.13	2.78	1.33	1.97 (63)	2.76	1.48	2.05 (65)
6.25	4.93	3.87	4.38 (70)	5.30	3.86	4.54 (73)
12.5	10.4	7.95	9.12 (73)	10.6	7.93	9.20 (74)
25.0	14.6	13.7	14.1 (56)	22.7	16.1	19.2 (77)
50.0	21.5	19.9	20.7 (41)	48.2	40.0	44.0 (88)
100	24.8	26.0	25.4 (25)	90.7	91.1	90.9 (91)

* values in parentheses represent percent of nominal concentrations

 

Table 2: Mean cell density of Pseudokirchneriella subcapitata exposure to Thiophanate-methyl

Mean measured	Mean Cell Concentration (x 10^4cells/mL)
concentration	after
[mg a.s./L]	0 h	24 h	48 h	72 h	96 h
Control	1.05	3.22	26.0	96.9	154
Solvent control	1.01	3.79	25.8	91.8	152
0.780	1.03	3.70	26.6	97.5	157
1.97	1.06	3.22	22.4	92.3	155
4.38	1.04	2.71	18.8	84.6	145
9.12	1.06	2.13	12.4	51.0	98.0
14.1	1.07	2.70	11.5	36.5	74.7
20.7	1.12	2.08	8.11	32.0	67.5
25.4	1.18	2.00	6.97	26.2	50.4

 

Table 3: Yield of Pseudokirchneriella subcapitata after exposure to Thiophanate methyl

Mean measured	Yield [x 10^4cells/mL] (percent inhibition)
concentration	after
[mg a.s./L]	24 h	48 h	72 h
Solvent control ^1	2.78	24.8	90.9
0.780	2.75 (1.1) *	25.6 (-3.4) *	96.5 (-6.2)
1.97	2.16 (22.2) *	21.3 (13.8) *	91.3 (-0.5)
4.38	1.68 (39.6) *	17.7 (28.4) *	83.6 (8.0)
9.12	1.07 (61.6) *	11.3 (54.4) *	49.9 (45.1) **
14.1	1.64 (41.1) *	11.1 (55.2) *	35.4 (61.0) **
20.7	-2.46 (189) *	3.57 (85.6) *	27.5 (69.8) **
25.4	0.823 (70.4) *	5.79 (76.6) *	25.1 (72.4) **

Values in parentheses represent percent inhibition compared to solvent control; negative values indicate a higher growth rate in treatment group compared to solvent control

¹ Inhibition of yield was compared to the solvent control as there were not statistically significant differences between the water control and the solvent control

* statistically significantly different compared to solvent control (p < 0.05, Bonferroni-Holm U-test)

** statistically significantly different compared to solvent control (p < 0.05, Williams t-test)

 

Table 4: Growth rate of Pseudokirchneriella subcapitata after exposure to Thiophanate-methyl

Mean measured	Growth rate [x 10^4cells/mL/d] (percent inhibition)
concentration	after
[mg a.s./L]	24 h	48 h	72 h
Solvent control ^1	1.32	1.62	1.50
0.780	1.30 (1.6) *	1.63 (-0.5) *	1.52 (-0.9) *
1.97	1.11 (15.6) *	1.53 (5.6) *	1.49 (1.0) *
4.38	0.962 (27.1) *	1.45 (10.6) *	1.47 (2.4) *
9.12	0.684 (48.2) *	1.22 (24.7) *	1.28 (14.7) *
14.1	0.930 (29.5) *	1.21 (25.3) *	1.17 (21.9) *
20.7	-0.151 (112) *	0.604 (62.7) *	0.862 (42.6) *
25.4	0.526 (60.1) *	0.888 (45.1) *	1.03 (31.4) *

Values in parentheses represent percent inhibition compared to control; negative values indicate a higher growth rate in treatment group compared to control

¹ Inhibition of growth rate was compared to the solvent control as there were not statistically significant differences between the water control and the solvent control

* statistically significantly different compared to control (p < 0.05, Bonferroni-Holm U-test)

 

Table 5: EC_x, LOEC and NOEC values for growth rate and yield of Pseudokirchneriella subcapitata after 72 hours of exposure to Thiophanate-methyl in a static test system

Function	EC10 [mg a.s./L]	EC20 [mg a.s./L]	EC50 [mg a.s./L]	LOEC [mg a.s./L]	NOEC [mg a.s./L]
Toxicity endpoints after 72 hours
Yield	3.13 (1.89 - 4.28)	4.93 (3.43 - 6.22)	11.8 (10.1 - 13.6)	9.12	4.38
Growth rate	6.74 (1.00 - 10.5)	12.1 (5.14 - 16.1)	37.2 (25.6 - 169)	n.d.	n.d.

 

Table 6: Comparison of EC_x, LOEC and NOEC values for yield and growth rate of Pseudokirchneriella subcapitata after 72 hours of exposure to Thiophanate-methyl in a static test system as presented in the study report with the re-calculated endpoints

	Endpoints as presented in original report (Saito, 2002)		Re-calculated endpoints
Yield^1
EC10 [mg a.s./L]		n.r.	3.13
EC20 [mg a.s./L]		n.r.	4.93
EC50 [mg a.s./L]		12	11.8
LOEC [mg a.s./L]		9.12	9.12
NOEC [mg a.s./L]		4.38	4.38
Growth rate
EC10 [mg a.s./L]		n.r.		6.74
EC20 [mg a.s./L]		n.r.		12.1
EC50 [mg a.s./L]		> 25.4		37.2
LOEC [mg a.s./L]		14.1		*
NOEC [mg a.s./L]		9.12		*

 ¹reported as area under the growth curve (biomass) in the study report by Saito (2002)

n.r. not reported

* could not be determined due to mathematical reasons or inappropriate data

Table 7: Relevant toxicity endpoints based on growth rate of Pseudokirchneriella subcapitata exposed to Thiophanate-methyl for 72 hours in a static test system to be used for the aquatic risk assessment 

Growth Function	EC10 [mg a.s./L]	EC20 [mg a.s./L]	EC50 [mg a.s./L]
Growth rate	6.74	12.1	37.2

Validity criteria fulfilled:

yes

Conclusions:

After the re-evaluation of the experimental results, the EC10, EC20 and EC50 values of the test item based on yield after 72 hours based on mean measured concentrations were 3.13, 4.93 and 11.8 mg/L, respectively. The EC10, EC20 and EC50 values of the test item based on growth after 72 hours based on mean measured concentrations were 6.74, 12.1 and 37.2 mg/L, respectively. The NOEC for yield after 72 h was 4.38 mg/L.

Executive summary:

The toxicity of the test item on the growth of the unicellular green alga, Pseudokirchneriella subcapitata during exposure periods of 72 and 96 hours under continuously shaking culturing / method was estimated in a method according to OECD 201. Since water solubility of the test substance is very small, OECD medium contained acetone at the rate of 0.1 mL/L was used.

The overall mean measured concentrations during the experiment for the test item at the nominal concentrations of 1.56, 3.13, 6.25,12.5, 25.0, 50.0 and 100 mg/L were 0.780, 1.97, 4.38, 9.12, 14.1 20.7 and 25.4 mg/L respectively. These values were 50, 63, 70, 73, 56, 41 and 25 % of the nominal test item concentrations. This phenomenon was considered to be due to incomplete dissolution resulted from lower water solubility of the test substance in the higher concentration and/or adsorption of the test substance on the wall of the glass ware and/or degradation. As the values were less than 80 % of the nominal concentrations estimation of the toxicity of the test item to P. subcapitata was performed based on the mean concentrations measured. According to the old OECD guideline from 1984, the study is considered valid if the cell growth in the control increased from the initial density by more than 16 times after 72 hours of growth.

The following endpoints were obtained:

Biomass: Based on mean measured concentrations, the 72-hour EbC50 value was determined to be 12 mg/L. The No-Observed-Effect Concentration (NOEC) was determined to be 4.38 mg/L.

Growth rate: Based on mean measured concentrations, the ErC50 value for the periods 24 - 48 hours and 24 - 72 hours was determined to be > 25.4 mg/L. The No-Observed-Effect Concentration (NOEC) for the periods 24 - 48 hours and 24 - 72 hours was determined to be 9.12 mg/L.

In 2006 and 2011, the OECD 201 guideline was revised, including additional validity criteria and the additional endpoint of yield. Therefore, the study was re-evaluated with the statistic programme ToxRat^® Professional (version 2.10) in order to see whether the study is still valid and complies with current guideline requirements with regard to other parameters (e.g. test conditions, cell number and pH values). In preparation of the renewal of the test item according to Annex I of Regulation (EC) No 1107/2009, the validity of the above available algal toxicity study with the test item was checked.

The new validity criteria as stated in the current OECD guideline are as follows: the cell growth in the control must increase from the initial density (1.0 x 10⁴ cells/mL) by more than 16 times after 72 hours of growth (actual increase 92.3 times); the mean coefficient of variation (CV) for section-by-section specific growth rates (day 0 to 1, 1 to 2 and 2 to 3) in the control replicates should not exceed 35 % (actual: 33.9 %). The CV for the average growth rate of the control for the entire test period (0- to 72-hour growth rate) should not exceed 7 % (actual 1.8 %). All validity criteria listed above were met.

The re-evaluation of the study (necessary due to an update of the guideline requirements) with the statistic programme ToxRat® Professional (version 2.10) showed that the study results accurately reflect the sensitivity of Pseudokirchneriella subcapitata to the test item under testing conditions as the concentration response was well-defined and allowed a clear definition of the EC values. The EyC10, EyC20 and EyC50 after 72 hours based on mean measured concentrations were 3.13, 4.93 and 11.8 mg/L, respectively. The ErC10, ErC20 and ErC50 after 72 hours based on mean measured concentrations were 6.74, 12.1 and 37.2 mg/L, respectively. The NOEC for yield after 72 h was 4.38 mg/L.

Description of key information

After the re-evaluation of the experimental results, the EC₁₀, EC₂₀ and EC₅₀ values of the test item based on yield after 72 hours based on mean measured concentrations were 3.13, 4.93 and 11.8 mg/L, respectively. The EC₁₀, EC₂₀ and EC₅₀ values of the test item based on growth after 72 hours based on mean measured concentrations were 6.74, 12.1 and 37.2 mg/L, respectively. The NOEC for yield after 72 h was 4.38 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

37.2 mg/L

EC10 or NOEC for freshwater algae:

6.74 mg/L

Additional information

The toxicity of the test item on the growth of the unicellular green alga, Pseudokirchneriella subcapitata during exposure periods of 72 and 96 hours under continuously shaking culturing / method was estimated in a method according to OECD 201. Since water solubility of the test substance is very small, OECD medium contained acetone at the rate of 0.1 mL/L was used.

The overall mean measured concentrations during the experiment for the test item at the nominal concentrations of 1.56, 3.13, 6.25,12.5, 25.0, 50.0 and 100 mg/L were 0.780, 1.97, 4.38, 9.12, 14.1 20.7 and 25.4 mg/L respectively. These values were 50, 63, 70, 73, 56, 41 and 25 % of the nominal test item concentrations. This phenomenon was considered to be due to incomplete dissolution resulted from lower water solubility of the test substance in the higher concentration and/or adsorption of the test substance on the wall of the glass ware and/or degradation. As the values were less than 80 % of the nominal concentrations estimation of the toxicity of the test item to P. subcapitata was performed based on the mean concentrations measured. According to the old OECD guideline from 1984, the study is considered valid if the cell growth in the control increased from the initial density by more than 16 times after 72 hours of growth.

The following endpoints were obtained:

Biomass: Based on mean measured concentrations, the 72-hour EbC50 value was determined to be 12 mg/L. The No-Observed-Effect Concentration (NOEC) was determined to be 4.38 mg/L.

Growth rate: Based on mean measured concentrations, the ErC50 value for the periods 24 - 48 hours and 24 - 72 hours was determined to be > 25.4 mg/L. The No-Observed-Effect Concentration (NOEC) for the periods 24 - 48 hours and 24 - 72 hours was determined to be 9.12 mg/L.

In 2006 and 2011, the OECD 201 guideline was revised, including additional validity criteria and the additional endpoint of yield. Therefore, the study was re-evaluated with the statistic programme ToxRat^® Professional (version 2.10) in order to see whether the study is still valid and complies with current guideline requirements with regard to other parameters (e.g. test conditions, cell number and pH values). In preparation of the renewal of the test item according to Annex I of Regulation (EC) No 1107/2009, the validity of the above available algal toxicity study with the test item was checked.

The new validity criteria as stated in the current OECD guideline are as follows: the cell growth in the control must increase from the initial density (1.0 x 10⁴ cells/mL) by more than 16 times after 72 hours of growth (actual increase 92.3 times); the mean coefficient of variation (CV) for section-by-section specific growth rates (day 0 to 1, 1 to 2 and 2 to 3) in the control replicates should not exceed 35 % (actual: 33.9 %). The CV for the average growth rate of the control for the entire test period (0- to 72-hour growth rate) should not exceed 7 % (actual 1.8 %). All validity criteria listed above were met.

The re-evaluation of the study (necessary due to an update of the guideline requirements) with the statistic programme ToxRat® Professional (version 2.10) showed that the study results accurately reflect the sensitivity of Pseudokirchneriella subcapitata to the test item under testing conditions as the concentration response was well-defined and allowed a clear definition of the EC values. The EyC10, EyC20 and EyC50 after 72 hours based on mean measured concentrations were 3.13, 4.93 and 11.8 mg/L, respectively. The ErC10, ErC20 and ErC50 after 72 hours based on mean measured concentrations were 6.74, 12.1 and 37.2 mg/L, respectively. The NOEC for yield after 72 h was 4.38 mg/L.