Thiophanate-methyl

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-03-22 to 1993-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: Protocol for Conducting a Flow-Through Acute Toxicity Test with Rainbow Trout (O ncorhynchus mykiss) Following FIFRA Guideline 72-1

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA. 1975. Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphib ians. Ecological Research Series (EPA-660/3-75-009).

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Prior to test initiation, a super-stock (14C) solution was first prepared by dissolving the entire amount of radiolabeled test material received (33.7 mg) with DMF to a volume of 100 mL. Triplicate assay determined that the concentration of the super-stock solution was 0.344 mg/mL. A 14C-Thiophanate-methyl stock solution was then prepared by adding 83.894 g (80.496 g as A.I.) of non-radiolabeled material with 68.1 mL of the super-stock solution (0.344 mg/mL). This solution was then diluted with DMF to a volume of 2000 mL resulting in a final stock solution concentration of 40.26 mg A.I. /mL. A second 14C-stock solution was prepared on test day 3 by adding 20.974 g (20.125 g as A.I.) of non-radiolabeled test material with 17.0 mL of the super-stock solution and then diluting with DMF to a total volume of 500 mL. Both stock solutions were stirred and sonicated to aid in the solubilization of the test material. Resultant stock solutions were observed to be amber in color and were transferred to glass bottles for use on the diluter system.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source: Spring Creek Trout Hatchery, a commercial supplier located in Lewistown, Montana, US.
- Length at study initiation: 3.7 (3.0 – 4.8) cm
- Weight at study initiation: 0.47 (0.28 - 0.75) g

ACCLIMATION
- Acclimation period: 14 days prior to testing
- Acclimation conditions: same as test
- Type and amount of food during acclimation: dry commercial pelleted food
- Feeding frequency during acclimation: ad libitum
- Health during acclimation: No mortality was observed in the test fish population during the acclimatisation period.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

25 - 26 mg/L, as CaCO3

Test temperature:

12 ± 1 °C

pH:

6.9 - 7.0

Conductivity:

100 - 110 µmhos/cm

Nominal and measured concentrations:

Nominal concentration 2.6, 4.3, 7.2 and 12 and 20 mg A.I./L
Mean measured concentrations averaged 103 % of the nominal concentrations and defined the treatment levels as 2.4, 4.7, 7.6, 13 and 20 mg A.I./L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Harvard Peristaltic apparatus pump
-Fill volume: 15 L
- Type of flow-through: peristaltic diluter
- Renewal rate of test solution: 500 mL per cycle
- No. of organisms per vessel: 10
- Biomass loading rate: 0.073 g of biomass per liter

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Total organic carbon: 1.5 mg/L
- Culture medium different from test medium: same
- Intervals of water quality measurement: once daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: 22-100 foot-candles

EFFECT PARAMETERS MEASURED: the fish were examined at test termination for sublethal effects and lethal effects

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.67
- Range finding study: yes
- Test concentrations: 5.2, 8.6, 14, 24, and 40 mg A.I./L and a dilution water control
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: LC50 value estimated by nonlinear interpolation; 95 % confidence interval calculated by binomial probability: (7.6 - 13)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence interval calculated by probit analysis: (12 - 15)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 20 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 20 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

At test termination (96-hours), 100 % and 75 % mortality was observed among fish exposed to the two highest mean measured concentrations tested (20 and 13 mg A.I./L, respectively). No mortality was observed among fish exposed to the remaining concentrations tested (2.4, 4.7, and 7.6 mg A.I./L). Sublethal effects (e.g., loss of equilibrium, erratic swimming) were observed among all of the surviving fish exposed to the 13 mg A.I./L test concentration, among several of the surviving fish exposed to the 7.6 mg A.I./L test concentration, and among one fish exposed to the 4.7 mg A.I./L test concentration.

Results with reference substance (positive control):

no information

Sublethal observations / clinical signs:

Table1: Test Results

LC50 (mg/L)^ab				No-Observed- Effect Concentration Through 96 Hours (mg/L)^a
24-Hour^c	48-Hour^c	72-Hour^d	96-Hour^e
> 20	> 20	14 (12-15)	11 (7.6-13)	2.4

^a Based on mean measured concentrations of Thiophanate-methyl (as active ingredient).

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

^c LC50 value empirically estimated to be greater than the highest mean measured concentration tested. 95 % confidence intervals could not be calculated.

^d LC50 value and 95 % confidence interval calculated by probit analysis.

^e LC50 value estimated by nonlinear interpolation; 95% confidence interval

 

Table 2: Concentrations of 14C-Thiophanate-methyl measured in replicate (A,B) test solutions during the 96-hour flow-through exposure of rainbow trout (Oncorhynchus my kiss).

Nominal Concentration (mg/L)	0-Hour Measured Concentration (mg/L)		96-Hour Measured Concentration (mg/L)		Mean Measured Concentration^a (mg/L)
	A	B	B	A
Control	< 0.36	< 0.36	< 0.36	< 0.36
Solvent Control	<0.36	< 0.36	< 0.36	<0.36
2.6	2.4	2.4	2.3	2.4	2.4 (0.050)
4.3	4.6	4.7	5.1	4.3	4.7 (0.33)
7.2	7.1	7.9	7.5	7.9	7.6 (0.38)
12	13	13	13	13	13 (0.076)
20	20	21	20	20	20 (0.57)
QC #1^b	5.95 (5.64)c		5.97 (5.64)
QC #2	6.70 (6.44)		6.43 (6.44)
QC #3	7.58 (7.25)		7.56 (7.25)

^a Mean measured concentrations are presented with the standard deviations in parentheses and were calculated using the unrounded analytical results 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 mortalities and observations made during the 96-hour flow-through exposure of rainbow trout (Oncorhynchus mykiss) to Thiophanate-methyl.

Cumulative Mortality (%)
Mean Measured Concentration (mg/L)	24-Hour			48-Hour			72-Hour			96-Hour
	A	B	Mean	A	B	Mean	A	B	Mean	A	B	Mean
Control	0	0	0	0	0	0	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0	0	0	0	0	0	0
2.4	0	0	0	0	0	0	0	0	0	0	0	0
4.7	0	0	0^f	0	0	0^f	0	0	0^f	0	0	0^f
7.6	0	0	0^be	0	0	0^bel	0	0	0^bgj	0	0	0^bck
13	10	0	5^bcd	20	0	10^bgh	40	40	40^a	70	80	75^a
20	0	10	5^a	40	50	45^a	90	100	95^a	100	100	100

^a All of the surviving fish exhibited complete loss of equilibrium.

^b Several of the surviving fish exhibited complete loss of equilibrium.

^c Several of the surviving fish exhibited partial loss of equilibrium.

^d One of the surviving fish was at the surface of the test solution.

^e One of the surviving fish exhibited partial loss of equilibrium.

^f One of the surviving fish exhibited complete loss of equilibrium.

^g One of the surviving fish was lethargic.

^h Two of the surviving fish exhibited darkened pigmentation and were lethargic.

ⁱ Several of the surviving fish were observed to be lethargic.

^j Two of the surviving fish exhibited partial loss of equilibrium.

^k Several of the surviving fish exhibited erratic swimming behavior.

Validity criteria fulfilled:

yes

Conclusions:

The No-Observed-Effect Concentration (NOEC) established during this study was 2.4 mg/L and the 96-hour LC50 was determined to be 11 mg/L.

Executive summary:

The purpose of this study was to estimate the acute toxicity (LC₅₀) of the test item to rainbow trout (Oncorhynchus mykiss) under flow-through conditions. The LC₅₀ is defined as the concentration of the test material in dilution water which causes mortality of 50 % in the exposed test population after a fixed period of time. Twenty organisms (ten per replicate) were exposed in duplicate test aquaria to each of five concentrations of the test item, a solvent control (dimethylformamide, DMF), and a dilution water control for 96-hours. The test solutions were prepared with a combination of non-radiolabeled and radiolabeled test material. During the test, nominal concentrations of 2.6, 4.3, 7.2, 12 and 20 mg/L were maintained by introducing approximately 6.4 aquarium volumes per day of newly prepared test solution via an intermittent-flow proportional diluter apparatus. Each replicate solution was sampled and analyzed for the test item concentration at 0-hour (test initiation) and 96-hours (test termination) of exposure. Based on the results of these analyses, the mean measured exposure concentrations were defined as 2.4, 4.7, 7.6, 13 and 20 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, undissolved test material (e.g., precipitate, surface film) was observed in the solution contained within the mixing chamber of the diluter system. No undissolved test material was observed in the diluter's chemical cell solutions or the splitter solutions. Exposure solutions were observed to be clear and colorless and contained no visible sign of undissolved test material (e.g., precipitate).

At test termination (96-hours), 100 % and 75 % mortality was observed among fish exposed to the two highest mean measured concentrations tested (20 and 13 mg/L, respectively). No mortality was observed among fish exposed to the remaining concentrations tested (2.4, 4.7, and 7.6 mg/L). Sublethal effects (e.g., loss of equilibrium, erratic swimming) were observed among all of the surviving fish exposed to the 13 mg/L test concentration, among several of the surviving fish exposed to the 7.6 mg/L test concentration, and among one fish exposed to the 4.7 mg/L test concentration. The No-Observed-Effect Concentration (NOEC) established during this study was 2.4 mg/L and the 96-hour LC₅₀ was determined to be 11 mg/L (95 % confidence interval of 7.6 - 13).

 

Additional remark: Based on these results the test item is considered to be slightly toxic to rainbow trout.

Description of key information

Short-term toxicity to rainbow trout (Oncorhynchus mykiss) in flow through system (EPA 660/3/75/009 and FIFRA 72-1) ref. Bettencourt 1993

NOEC = 2.4 mg/L

LC₅₀ = 11 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

11 mg/L

Additional information

The following endpoints have been used to address the short-term toxicity of the test item to fish. The endpoints from the study performed according to US EPA 660/3/75/009 and FIFRA 72-1 under GLP is used for risk assessment.

 

Short-term toxicity to rainbow trout (Oncorhynchus mykiss) in flow through system

The No-Observed-Effect Concentration (NOEC) established during this study was 2.4 mg/L and the 96-hour LC₅₀ was determined to be 11 mg/L.

 

Two additional studies testing the acute toxicity of the test item to fish were conducted and used as supporting information to cover the endpoint.

 

Short-term reproduction assay in fathead minnow (Pimephales promeias) in flow through system

This study was conducted under flow through conditions following the OECD guideline 229 under GLP. Reproduction in terms of fecundity as well as secondary sex characteristics was recorded. The nominal test concentrations were 0.1, 0.316 and 1 mg/L. Bacterial infection was detected in the fish, which induced alterations of the fish gonadal tissue. Single fish expressed clinical signs which could be related to this infection. However, no dose related increase of disease was detected. The bacteria infection can thus be considered as a background stressor, which affects the fish without clear link to a test item dose and became obvious by the expression of clinical signs.

The evaluation of the endocrine related endpoints revealed no dose-related negative impact of the test item. No effect on reproduction could be detected in the test. The measurement of the biomarker vitellogenin as well as the assessment of the endocrine modulated secondary sex characteristics revealed no dose related negative response to test item exposure.

An effect on the maturity index of males could be observed in the two highest treatment conditions. However, due to alterations of the fish tissue which were linked to the bacteria disease, the interpretation of potential endocrine effects on the tissue level was impeded.

Nevertheless, no exposure related negative trend of pathological findings could be detected.

To conclude, a relevant endocrine disrupting potential of the test item could not be detected in this study.

 

Short-term reproduction assay in fathead minnow (Pimephales promeias) with aged test item, (Static conditions in a water sediment system)

The study was conducted in accordance to the OECD guideline 229, however with the deviation of conduction under static conditions. Reproduction in terms of fecundity and secondary sex characteristics were recorded. Furthermore, blood plasma samples were taken from the test fish and measured for vitellogenin. Finally, a histopathological evaluation of the fish gonads was conducted.

Aging of the test item was performed in order to allow the conversion of the test item to the main metabolite carbendazim (MBC). MBC peak concentrations of 0.0097, 0.0246, and 0.06 mg/L (119.9 % to 121.5 % of the target concentration) were achieved. The TWAs of MBC were calculated to be 0.009, 0.0228, and 0.0561 mg MBC/L.

Bacterial infection was detected in the majority of fish, irrespective of the treatment level and also in controls, and induced alterations in the fish tissue. Expression of clinical signs and altered behaviour (e.g. swimming behaviour) of fish during the study could be related to this infection. However, no dose related increase of toxic symptoms due to bacterial infection was detected.

Evaluation of the endocrine-related endpoints revealed a concentration-related negative impact on the secondary sexual characteristics (SSCs) of males during the in-life phase of the study. A statistically significant decrease of the formation of mating colour and nape pad was observed by visual observations in the time course of the study, also a reduction of territorial aggressiveness, in the highest treatment level of 0.428 mg test item 500 SC/L (0.06 mg MBC/L (peak); 0.0561 mg MBC/L (TWA)). Effects on SSCs vanished over the time, indicating an only delayed and less pronounced reproductive activity, but not a complete absence. SSCs of females (i.e. formation of an ovipositor) were not affected.

A further effect related to the reproductive activity was a statistically significant reduction of spawning days in the highest treatment level. Additionally, the cumulative egg no per replicate was reduced compared to the control, however not statistically significant. The measurement of the biomarker VTG revealed no dose related negative response to test item exposure. Due to alterations of the fish tissue, which were linked to the bacteria disease, the interpretation of potential endocrine effects on the tissue level was impeded. Furthermore, the interpretation of reproduction-related data is also impaired by the bacterial infection. Nevertheless, no exposure related trend of pathological findings could be detected, as no difference in the severity of the infection was detected between controls and treatments, and thus, can be considered as background stressor. To add, an impact of the bacterial infection on the reproductive performance is not very likely, as the groups of the controls and the two lower treatments levels displayed very active spawning despite of the massive bacterial infection.

In conclusion, a concentration-dependent negative impact on the endocrine-related endpoints SSCs of male fish and the number of spawning events was detected. However, effects on SSCs of males and the number of spawning events might be influenced by the number of introduced males, that unintentionally diverged from the intended number of 2 males/replicates (4 males in vessel 3/1; 3 males in vessels 3/3 and 3/4). Effects on primary endocrine endpoints, i.e. VTG content, egg number/female/day, and tubercle scoring, were not observed.

Based on the most sensitive endpoints, the highest concentration showing no effect (NOEC) in this study was determined to be: 0.171 mg test item 500 SC/L corresponding to 0.0246 mg carbendazim/L (peak); 0.02277 mg carbendazim/L (TWA).