Disulfiram

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 Jan - 06 Apr 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Main test was technically not feasible since no analytical measurements could be performed

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

adopted July 2013

Deviations:

not applicable

Remarks:

No main test was performed

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Analytical verification was performed but was not sensitive enough for the main test

Details on sampling:

- Concentrations: Solvent control and each test concentration
- Sampling method: Samples (ca 8 mL) were taken for analysis prior to exposure of the eggs and on Day 0 pre-hatch (start of exposure period) and Days 0, 2, 6, 9 and 14 post-hatch. At each sampling occasion, triplicate samples were taken. One sample was used for chemical analysis, and two were retained as ‘back up’ samples should further analysis be required.

Vehicle:

yes

Remarks:

dimethylformamide (DMF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A 1 mg/mL solvent stock solution was prepared by dissolving ca 25 mg of the test substance in 25 mL of DMF. Serial dilutions were prepared, in DMF, from this solvent stock solution to give further solvent stock solutions of 0.1 and 0.01 mg/mL. Although serial dilutions are generally avoided, in this case this approach was necessary due to the concentrations required.
The 0.01, 0.1 and 1 mg/mL solvent stock solutions were each separately dosed at a rate of 0.12 mL/hour to a dilution water flow rate of 20 ± 10% mL/minute to give the 1.0, 10 and 100 µg/L test concentrations, respectively. A solvent control was prepared in a similar manner by dosing DMF at a rate of 0.12 mL/hour to a dilution water flow rate of 20 ± 10% mL/minute.
- Solvent Control: Yes

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: In-house breeding system
- Age at study initiation (mean and range, SD): Eggs were used

- Method of breeding:
Viable fish eggs were obtained from breeding groups of Pimephales promelas. Prior to initiation of the test, the stage of egg development was established. The eggs were added to the test system, typically before the first cleavage of the blastodisc or within 24 hours of fertilisation.
The breeding groups in the breeding system were held in a temperature-controlled room under artificial light (with a 16 hour light:8 hour dark photo-period with a ca 30 minute dawn/dusk period) in holding tanks appropriate to their size, under continuous water renewal (flow-through) conditions.

POST-HATCH FEEDING
- Start date: Developing larvae waq fed with 24-old shrimp; from day 6 the larvae was fedd with 48-old shrimp
- Food type: The breeding groups of Pimephales promelas were fed with 24 to 48 hour old brine shrimp (Artemia salinis) nauplii and also with Tetramin® flake food
- Amount: The quantities of feeding was dictated by the size of the fish.
- Frequency: The breeding groups of Pimephales promelas were fed 2 to 4 times daily

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Hardness:

CaCO3: 27.0 mg/L

Test temperature:

23.8 - 26.3 °C

pH:

6.93 - 7.68

Dissolved oxygen:

76 - 103.3% ASV

Nominal and measured concentrations:

1.0, 10 and 100 µg/L (nominal)

Details on test conditions:

RANGE-FINDING STUDY
TEST SYSTEM
Test vessel:
- Egg incubation chamber: The chambers used in the test were 17 cm long glass cylinders (6.5 cm internal diameter) with a 0.5 - 1.0 mm non-reactive mesh attached to one end (bottom)
- Size of vessel: 3.5 L aquariums
- Type: open
- Material, size, headspace, fill volume: 3.5 L constructed glass aquariums, 3 L volume (0.5 L headspace)
- Type of flow-through: peristaltic pump was used
- Renewal rate of test solution: 0.12 mL/hour to a dilution water flow rate of 20 ± 10% mL/minute. The flow rates used represented an approximate media exchange rate of 10 volumes per 24 hours per test vessel.
- No. of organisms per vessel: 10 eggs per treatment and solvent control group
- No. of vessels per concentration: 1
- No. of vessels per vehicle control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water: The water used was laboratory treated mains supply. The water was pumped to the laboratory through an activated carbon filter. pH 7.4; BOD+ATZ (5 days): <1 mg/L; Alkalinity as CaCO3: 27.0 mg/L
- Source/preparation of dilution water: Process water (blended spring and reverse osmosis waters); according to guideline
- Culture medium different from test medium: No
- Intervals of water quality measurement: At experimental start (pre-hatched), Day 2 (post-hatched), Day 9 (post hatched), Day 16 (post hatched)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED: Sublethal effects, fish hatchability, survival, and growth (length and dry weight- all surviving fish on Day 14)

POST-HATCH DETAILS
- Begin of post-hatch period: Day 6

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 (range-finding study)

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: See Remark in section: "Any other information on results incl tables"

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

> 1 - < 10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks on result:

other: post-hatch survival

Remarks:

See remark in section: "Any other information on results incl tables"

Details on results:

- Observed effects at each concentration for each observation time: see Table [#?]
- Concentrations that produce lethal or other effects: [describe and attach graph showing effects with respect
to time]
- Cumulative mortality at each concentration and for each recommended observation time if possible:
- Mortality in the controls:
- Behavioural observation of the fish:
- Effect concentrations exceeding solubility of substance in test medium:
- Incidents in the course of the test which might have influenced the results:

Remark regarding key results:

Based upon empirical evaluation of the range-finding data, the NOEC and LOEC for hatching success could be assumed to be at 100 and >100 µg/L, respectively, based on nominal test concentrations. The NOEC and LOEC concentrations for Day 14 post-hatch survival are expected to be in the range of 1.0 and 10 µg/L, respectively, based on nominal concentrations.

Biological Results:
Table 1: Hatching success and fish larval survival during the range-finding test

Nominal concentration (µg/L)	Number of eggs added at start of test	Number of hatched larvae	Percentage of hatched larvae (hatching success)	Number of larvae surviving at 14 days post-hatch	Percentage of larvae surviving at 14 days post-hatch (post-hatch survival)
Solvent control	10	7	70	6	86
1.0	10	9	90	8	89
10	10	10	100	0	0
100	10	9	90	0	0

All larvae at 100 µg/L died on day of hatching;

All larvae at 10 µg/L died by Day 1 post-hatch

Table 2: Hatching Success 

Nominal Concentration [µg/L]	Hatching success [%]
Solvent control	70
1.0	90
10	100
100	90

  

Table 3: Post-hatched survival 

Nominal Concentration [µg/L]	Day 14 post-hatch survival [%]
Control	86
1.0	89
10	0
100	0

 

Conclusion

The objective of the study was to define the lethal and sub-lethal effects of the test substance, TETD, on the early developmental and juvenile stages of the freshwater fish species,Pimephales promelas, in accordance with OECD Chemical Testing Guideline No. 210 Fish, Early Life Stage Toxicity Test (July 2013).

The range-finding test was conducted at nominal test concentrations of 1.0, 10 and 100 µg/L. A solvent control group was also included.

Based upon empirical evaluation of the range-finding data, the NOEC and LOEC for hatching success could be assumed to be at 100 and >100 µg/L, respectively, based on nominal test concentrations. The NOEC and LOEC concentrations for Day 14 post-hatch survival are expected to be in the range of 1.0 and 10 µg/L, respectively, based on nominal concentrations.

Chemical analysis during the range-finding test showed that nominal concentrations were not achieved in the test vessels.Although all larvae in the 10 and 100 µg/L test concentrations had died by Day 1 post-hatch, samples were taken from these concentrations throughout the test to monitor measured concentrations.

The analytical data indicated that there was a pervasive background concentration of the test substance present therefore measured concentrations in the test solutions were evaluated against the background level observed in the solvent control. In general, values observed for the 1.0 and 10 µg/L nominal test concentrations were indistinguishable from those observed for the solvent control. Measured concentrations were observed in the 100 µg/L test concentration which ranged from 6% to 50% of nominal value throughout the test.

The results from the range-finding test indicated that at least a nominal concentration range of 1 to 10 µg/L would be required for the definitive test with the NOEC being in the range of 1 µg/L. The proposed limit of quantification (LOQ) for the analytical method was 1 µg/L.

Despite extensive efforts, a reliable LOQ of <1 µg/L was not considered to be achievable: in order to concentrate the test substance, Liquid-Liquid extraction (LLE) with various solvents, as well as Solid-Phase Extraction (SPE) have been performed. However, the background contamination present in the media was also concentrated to the same extent. Hence, the fortified and unfortified samples could not be distinguished as the additional fortified concentration was small compared with the endogenous background. Efforts to eliminate the contamination, i.e. changing suppliers and types of solvent and reagent, eliminating certain types of rubber and plastic materials from the analytical process and regular checking and testing of materials, were also not able to permit measurement at concentration below 1 µg/L. The analytical method suffers from limited robustness, despite the use of isotopically labelled internal standards and high-resolution mass spectrometry.

As described above a pervasive background attributable to the test substance is observed throughout the test. The background concentration is significant at the proposed LOQ.This background signal inhibits an improvement of the LOQ below 1 µg/L. Beside thata high variabilityof recoveries from the quality control samples at nominal loading concentrations 20 – 150 µg/Lwas observedthroughout the range-finding test, which could not fulfil the acceptance criteria for analytical method validation.

According to the OECD 210 paragraph 4, a reliable analytical method should be available for the quantification of the chemical in the test solutions with known and reported accuracy and limit of quantification.

In conclusion it was considered technically not feasible and unethical to conduct a definitive test. Therefore the definitive test was cancelled.

Validity criteria fulfilled:

not applicable

Remarks:

Main test was technically not feasible since no analytical measurements could be performed, therefore validity criteria of the main test could not be reported

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP-Guideline study, minor restrictions in design and/or reporting but otherwise adequate for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The dilution water was a synthetic medium, Dutch Standard Water according to NPR 6507, containing 100 mg Na HCO3/L, 20 mg KHCO3/L, 200 mg CaCl2.2H2O/L and 180 mg MgSO4. 7H2O/L. The pH is approximately 8.2, the hardness 13 dH. The medium was aerated before use.

Test media of the water-soluble test stubstances were prepared by dissolving the test substance in the dilution water. For the poorly water-soluble substances, a stock solution in acetone or DMSO was made, from which a second stock solution in dilution water was prepared. As the water solubility of most test substances was unknown, the calculations are based on nominal concentrations. The test substances did not always solve well in the dilution water and formed dispersions. Where nominal concentrations exceed the water saturation values, the nominal cocnetrations should be interpreted accordingly. The pH, oxygen concentration and temperature were measured and adjusted if necessary, before the test animals were introduced in the medium.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Brachydanio rerio (new name Danio rerio) were obtained form a local aquarium retailer and kept in the laboratory at 24 °C, males and females in separate groups. They were bred at regular intervals: in the afternoon females were isolated in small tanks filled with a mesh bottom at 26 °C. After a few hours two males were introduced before the lights went off. In the morning after the lights were turned on, eggs were produced. Each batch of eggs was collected from the bottom of the tank by a small fishing net and transferred to a small volume of water. White and opaque eggs and empty shells were removed and the quality of the batch and their stage of development was assessed. For each test, eggs of only one female were used to insure a more homogeneous population of test eggs. On several occassions, test were repeated, enabling to compare the sensitivity of batches of different origin.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

10 d

Hardness:

200 mg CaCO3/l

Test temperature:

25°C

pH:

8.2

Dissolved oxygen:

Saturated

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 3.2, 10, 32, 100, 320 ug/L

Details on test conditions:

Test containers were polystyrene multi-dishes with 6 holes each containing 5 mL of test medium. Five to seven eggs were transferred to each hole by means of a glass tube equiped with a rubber bulb. Incubation started within 4 hours after spawning (gastrula stage). The eggs were incubated at 25 °C under artificial light during working hours. Daily observations were made on survival of eggs and larvae, hatching and malformations. Dead organisms were removed. The test was semi-static: the medium was renewed after two or three days. Oxygen concentration, pH and temperature were measured to confirm that test conditions were normal. The NOEC concentrations for survival, hatching and malformations were determined graphically.

Reference substance (positive control):

no

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

3.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

3.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

<= 10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: malformations

Results with reference substance (positive control):

Not applicable

Reference 3

Endpoint:

fish early-life stage toxicity

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Description of key information

Study is technically not feasible.

Additional information

Due to analytical problems, the main test for the chronic fish test was considered as technically not feasible. Thus the endpoint long term toxicity to fish has been waived. An OECD 210 range finding study was run but the main test was cancelled as a pervasive background attributable to the test substance was observed throughout the test. The effects was as such, that no clear analytical dose verification was possible for the chronic fish test. Available results from the range-finding test on fish indicated that at least a nominal concentration of 1 to 10 µg/L would be required for the definitive test with the NOEC being in the range of 1 µg/L, which is within the same range as the chronic Daphnia study. Due to the impossibility to conduct the chronic fish test with appropriate analytical verification, the PNEC for TETD was therefore derived based on the long-term daphnia study with an EC10 value of 0.00264 mg/L (measured concentration).