Thiophanate-methyl

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-04-08 to 1992-04-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Protocol for Conducting a Flow-Through Acute Toxicity Test with Water Fleas (Daphnia magna) Following FIFRA Guideline 72-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA- 54C/6-85, Standard Evaluation Procedures for Acute Toxicity Test for Freshwater Invertebrates.

Version / remarks:

June 1985

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Methods for Acute Toxicity Tests with Fish, Microinvertebrates, and Amphibians. Ecological Research Series (EPA-660/3-75-009). 61 pp.

Version / remarks:

1975

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Sampling procedures typically include syphoning (using silicone tubing) from the midpoint of the test container into graduated cylinders for volumes greater than 200 mL, and pipetting (using volumetric pipets) from the midpoint of the test container for sample volumes less than or equal to 200 mL. Triplicate (10.0 or 25.0 µL) aliquots were removed from each of the five stocks (A - E) for analysis by Liquid Scintillation Counting (LSC).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A 50 mg/mL 14C stock solution was prepared by first diluting the entire sample of radio-labeled test item (approximately 18 mg) with DMF to volume in a 50 mL flask. Based upon triplicate radiometric analyses and the specific activity of 20.4 mCi/mmole, the concentration of the resultant super-stock solution was determined to be 0.378 mg/mL. A 10.2786 g (10.0288 g as A.I.) sample of the non-radiolabeled test material was then mixed with 7.0 mL of the 0.378 mg/mL super-stock solution in a 200 mL volumetric flask and brought to volume with DMF. The concentration of the resultant 14C stock solution was 50 mg/mL (nominal).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: Springborn Laboratories, Inc., Wareham, Massachusetts, US

ACCLIMATION
- Acclimation period: 2 weeks prior to study initiation
- Acclimation conditions: same as test
- Type and amount of food: combination of a trout food suspension and a unicellular green algae (Ankistrodesmus falcatus)
- Feeding frequency: once daily
- Health during acclimation: < 4 % mortality in the culture population

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

(CaCO3) = 160 - 170 mg/L

Test temperature:

18 - 21 °C

pH:

8.1 - 8.2

Dissolved oxygen:

8.4 – 9.1 mg/L

Conductivity:

500 µmhos/cm

Nominal and measured concentrations:

Nominal concentration: 3.1, 5.2, 8.6, 14 and 24 mg/L
Mean Measured concentration: 4.2, 5.3, 9.5, 15 and 24 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967) and a set of 14 exposure vessels.
- Type of flow-through: peristaltic pump
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture / dilution water was prepared by fortifying Town of Wareham well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column and a carbon filter.
- Intervals of water quality measurement: 0 h, 24h and 48 h

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity: 30 - 50 footcandles

EFFECT PARAMETERS MEASURED: mobility

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 5.2, 8.6, 14, 24 and 40 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence interval calculated by moving average angle analysis: 4.4 - 6.3 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

< 4.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence interval calculated by probit analysis: 11 - 20 mg/L

Details on results:

Following 48 hours of exposure, immobilization of 100, 95 and 100 % was observed among daphnids exposed to the three highest mean measured concentrations tested (24, 15, and 9.5 mg/L). Immobilization of 50 and 20 % was observed among organisms exposed to the remaining concentrations tested (5.3 and 4.2 mg/L, respectively). In addition, sublethal effects (e.g., lethargy) were observed among all of the mobile daphnids exposed to the 15, 5.3 and 4.2 mg/L test concentrations.

Results with reference substance (positive control):

Not specified

Preliminary test

Prior to initiating the definitive study, a 48-hour preliminary range-finding test was conducted. During this 48-hour period, daphnids were exposed under flow-through conditions to nominal concentrations of the test item ranging from 40 - 5.2 mg/L. At test termination (48 hours), immobilization of 100 % was observed among daphnids exposed to the two highest concentrations tested (40 and 24 mg/L). Immobilization ranging from 0 - 5 % was observed among organisms exposed to the remaining concentrations tested (14, 8.6 and 5.2 mg/L). In addition, sublethal effects (e.g., lethargy) were observed among all mobile daphnids exposed to these treatment levels. Based on these results, nominal concentrations of 24, 14, 8.6, 5.2 and 3.1 mg/L were selected for the definitive study.

 

 

Table 1: Test Results

EC50 (mg A.l./L)^ab		No Observed Effect Concentration Through 48 Hours^a (mg/L)
24-Hour^c	48-Hour^d
14 (11 - 20)	5.4 (4.4 – 6.3)	< 4.2^e

^a Based on mean measured concentrations of 14C-Tbiophanate-methyi (as active ingredient).

^b Corresponding 95 % confidence interval is presented in parentheses.

^c EC50 value and 95 % confidence interval calculated by probit analysis.

^d EC50 value and 95 % confidence interval calculated by moving average angle analysis.

^e The lowest mean measured concentration tested.

 

Table 2: Concentrations of 14C-Thiophanate-methyl measured in replicate (A,B) test solutions during the 48-hour flow-through exposure of daphnids (Daphnia magna).

 

	Measured Concentration (mg/L)
Nominal concentration (mg/L)	0-Hour		48-Hour		Mean measured Concentration^a(mg/L)
	A	B	A	B
24	23	24	24	23	24 (0.60)
14	16	15	14	15	15 (0.60)
8.6	9.6	10	9.1	9.4	9.5 (0.40)
5.2	5.4	5.4	5.1	5.2	5.3 (0.20)
3.1	4.2	4.3	4.1	4.1	4.2 (0.10)
Solvent control	< 0.29	< 0.29	< 0.29	< 0.29
Contol	< 0.29	< 0.29	< 0.29	< 0.29
QC #1^b	2.53 (2.40)^c		23.6 (24.0)
QC #2	3.49 (3.20)		32.5 (32.0)
QC #3	4.26 (4.00)		40.7 (40.0)

^a Mean measured concentrations are presented with the standard deviation in parentheses and were calculated using the actual analytical value and not the rounded (two significant figures) values presented in this table.

^b QC = Quality Control sample.

^c Value in parentheses represents the nominal fortified concentration for the corresponding QC sample.

 

Table 3: Mean measured concentrations tested, corresponding cumulative percent of immobilized daphnids (Daphnia magna) and observations made during the 48-hour flow-through exposure to Thiophanate-methyl.

Mean Measured Concentration (mg/L)	Cumulative Percent Immobilized Organisms
	24-Hour			48-Hour
	A	B	Mean	A	B	Mean
24	60	60	60^a	100	100	100
16	80	40	60^b	100	90	95^e
9.5	40	60	50^c	100	100	100
5.3	20	0	10^c	60	40	50^e
4.2	0	0	0^d	30	10	20^e
Solvent control	0	0	0	0	0	0
Control	0	0	0	0	0	0

^a All of the mobile daphnids were observed to be lethargic.

^b Five of the mobile daphnids were observed to be lethargic.

^c Two of the mobile daphnids were observed to be lethargic.

^d One of the mobile daphnids was observed to be lethargic.

^e All of the mobile daphnids were observed to be lethargic and on the bottom of the test vessels.

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 value (95 % confidence interval) was calculated by moving average angle analysis to be 5.4 (4.4 - 6.3) mg/L. The NOEC established for this study was determined to be < 4.2 mg/L, the lowest mean measured concentration tested.

Executive summary:

The toxicity of the test item to Daphnia magna under flow through conditions was examined in a method according to US EPA – 54C/6-85 (1985) where the young daphnids were exposed to nominal concentrations of the test item. The EC50 is defined as the concentration of test material estimated to cause immobilization among 50 % of a test population at the stated exposure interval. Twenty organisms (ten per replicate) were exposed in duplicate test vessels to five concentrations of the test item, a solvent (dimethylformamide (DMF), CAS # 68-12-2) control and a dilution water control for 48 hours. During the test, nominal concentrations of 24, 14, 8.6, 5.2 and 3.1 mg/L were maintained in the exposure vessels by introducing approximately 6.0 test chamber volumes per day of newly prepared test solution via intermittent-flow proportional diluter apparatus. Each replicate solution was sampled and analyzed for Thiophanate-methyl at 0-hour (test initiation) and 48-hours (test termination) of the exposure period. Based on the results of these analyses, the mean measured exposure concentrations were defined as 24, 15, 9.5, 5.3 and 4.2 mg/L. Biological observations and observations of the physical characteristics of the exposure solutions were made and recorded at test initiation and every 24 hours thereafter until the test was terminated. Throughout the exposure period, no visible signs of undissolved test material (e.g., precipitate) was observed in either the diluter system or in the exposure solutions.

Following 48 hours of exposure, immobilization 100, 95 and 100 % was observed among daphnids exposed to the three highest mean measured concentrations tested (24, 15, and 9.5 mg/L). Immobilization of 50 and 20 % was observed among organisms exposed to the remaining concentrations tested (5.3 and 4.2 mg/L, respectively). In addition, sublethal effects (e.g., lethargy) were observed among all of the mobile daphnids exposed to the 15, 5.3 and 4.2 mg/L test concentrations. The 48-hour EC₅₀ value (95 % confidence interval) was calculated by moving average angle analysis to be 5.4 (4.4 - 6.3) mg/L. The No Observed Effect Concentration (NOEC) through 48-hours of exposure was established to be < 4.2 mg/L, the lowest mean measured concentration tested.

 

Additional remark: Based on this data the test item was considered to be a moderate toxicant to Daphnia magna.

.

Description of key information

The 48-hour EC₅₀ value (95% confidence interval) was calculated by moving average angle analysis to be 5.4 (4.4 - 6.3) mg/L. The NOEC established for this study was determined to be < 4.2 mg/L, the lowest mean measured concentration tested.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

5.4 mg/L

Additional information

The toxicity of the test item to Daphnia magna under flow through conditions was examined in a method according to US EPA – 54C/6-85 (1985) where the young daphnids were exposed to nominal concentrations of the test item. The EC₅₀ is defined as the concentration of test material estimated to cause immobilization among 50 % of a test population at the stated exposure interval. Twenty organisms (ten per replicate) were exposed in duplicate test vessels to five concentrations of the test item, a solvent (dimethylformamide (DMF), CAS # 68-12-2) control and a dilution water control for 48 hours. During the test, nominal concentrations of 24, 14, 8.6, 5.2 and 3.1 mg/L were maintained in the exposure vessels by introducing approximately 6.0 test chamber volumes per day of newly prepared test solution via intermittent-flow proportional diluter apparatus. Each replicate solution was sampled and analyzed for Thiophanate-methyl at 0-hour (test initiation) and 48-hours (test termination) of the exposure period. Based on the results of these analyses, the mean measured exposure concentrations were defined as 24, 15, 9.5, 5.3 and 4.2 mg/L. Biological observations and observations of the physical characteristics of the exposure solutions were made and recorded at test initiation and every 24 hours thereafter until the test was terminated. Throughout the exposure period, no visible signs of undissolved test material (e.g., precipitate) was observed in either the diluter system or in the exposure solutions.

Following 48 hours of exposure, immobilization 100, 95 and 100 % was observed among daphnids exposed to the three highest mean measured concentrations tested (24, 15, and 9.5 mg/L). Immobilization of 50 and 20 % was observed among organisms exposed to the remaining concentrations tested (5.3 and 4.2 mg/L, respectively). In addition, sublethal effects (e.g., lethargy) were observed among all of the mobile daphnids exposed to the 15, 5.3 and 4.2 mg/L test concentrations. The 48-hour EC₅₀ value (95 % confidence interval) was calculated by moving average angle analysis to be 5.4 (4.4 - 6.3) mg/L. The No Observed Effect Concentration (NOEC) through 48-hours of exposure was established to be < 4.2 mg/L, the lowest mean measured concentration tested.

 

Additional remark: Based on these data the test item is considered to be a moderate toxicant to Daphnia magna.