reaction mass of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2H -isothiazol-3-one

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Nov-12 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were collected from control (on day 35), lowest test concentration and highest test concentration (both on days 0, 7, 14, 21, 28 and 35 of the experiment).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared by weighing 71.0-73.2 mg of test item into 100 mL of dilution medium (nominal concentration of 0.704 mg test item/L). The test item was mixed into the water as homogeneously as possible by intense stirring. The stock solution was prepared every week.
For the test, the stock solution was delivered by the flow-through system to mixing vessels. The solutions were stirred using a magnetic stirrer. Test solutions were delivered directly from the mixing vessels to the test vessels using teflon tubing and a multichannel peristaltic pump set to deliver the desired volume. The table below describes the procedure for preparing test solutions in the flow-through test system depending on the concentration of test item:
Nominal concentration (µ test item/L) Code of stock solution Volume of dilution medium per run (mL*) Volume of stock solution per run (mL*) Number of strokes dilution medium per run (n**) Number of strokes stock solution per run (n**)
Control - 350 0 8.5 0.0
7.04 S 347 4 8.5 15.6
22.3 S 339 11 8.3 49.3
70.4 S 315 35 7.7 22.0
223 S 239 111 5.8 69.7
704 S 0 350 0.0 220.1
* nominal volume delivered to the mixing vessel of the flow through system
** Nominal numbers of strokes per run of the flow through system
Time (in seconds) for pumps to deliver the required volumes of solutions during one run (TR): 351s
Number of runs per 24 h: 24 (3600 s per run); November 2 to December 6, 2012
- Controls: blank control

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: fertilised eggs were collected from adult zebra-fish from in-house cultures, which have been maintained and bred at testing facilities since August 2011

HOLDING CONDITIONS AND FERTILISATION
- In the culture tanks, the fish were kept in reconstituted water (according to OECD 203) diluted with deionised water (1:1; v/v), and supplemented with 1% of artificial seawater. Fish were held in glass aquaria at 26 ± 2° C with a 12 h/12 h photoperiod. They were fed a combined diet of newly hatched nauplii of Artemia sp. and TetraMin.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Hardness:

161-164 mg CaCO3/L

Test temperature:

25.3-26 °C

pH:

6.8-7.7

Dissolved oxygen:

64-98% air saturation value

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal: control, 7.04, 22.3, 70.4, 223 and 704 µg test item/L, corresponding to
control, 1.13, 3.59, 11.3, 35.9 and 113 µg CMI/L
Measured: The recovery of measured concentrations at the highest concentration tested (113 µg CMI/L) ranged between 19-81% of nominal. At the lowest tested concentration, recoveries ranged from 4.3-79%. A mean measured concentration was calculated using measured concentrations in the lowest and highest test solutions of days 0, 7, 14, 21, 28 and 35. The total mean recovery over all measured samples was 40.9% of nominal CMI concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessels, diameter 14 cm, height 7 cm; vessels were fitted with a meshed overflow: outflow at: 5.2 cm
- Aeration: yes
- Type of flow-through: peristaltic pump. The flow rates were measured volumetrically at least 3 times per week. The volume delivered/measured was divided by the number of strokes. The variation of the nominal stroke-volume was calculated by dividing the measured flow (mL/stroke) by the nominal volume per stroke of the designated pump expressed in percentage of nominal values. Additionally, the distribution between the replicates was measured once per week
- Renewal rate of test solution (frequency/flow rate): five vessel volumes per vessel per day
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: maximum 1.026 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was used to dilute the test item and to keep the fish before and during the test, (OECD Guideline No. 203) mixed with deionised water (1:1; v/v), supplemented with artificial water. Local tap water was treated by reverse osmosis and ion-exchanger to prepare deionised water. The reconstituted water consists of deionised water and salts in the following final concentrations:
a) CaCl2 x 2H2O 294.0 mg/L
b) MgSO4 x 7H2O 123.0 mg/L
c) NaHCO3 64.8 mg/L
d) KCl 5.75 mg/L
The required amount of reconstituted water was prepared within four weeks before use. During storage reconstituted water was aerated.
pH: 7.4-7.9
Conductivity: 750-1068 µS/cm
Total hardness: 154-239 mg CaCO3/L

OTHER TEST CONDITIONS
- Photoperiod: 12 h light/12 hour dark cycle
- Light intensity: 721-955 lux

EFFECT PARAMETERS MEASURED: hatching success, mortality (post-hatch success), body abnormalities, behavioural abnormalities, length of the surviving fish and dry weight of the surviving

POST-HATCH DETAILS
- Begin of post-hatch period: Fish started hatching on day 3 after start of exposure. Hatching was finished on day 8 for all treatments
- Cumulative number of fish (values of 2 replicates):
Control 56 hatched fish
1.13 µg CMI/L 58 hatched fish
3.59 µg CMI/L 60 hatched fish
11.3 µg CMI/L 58 hatched fish
35.9 µg CMI/L 55 hatched fish
113 µg CMI/L 59 hatched fish

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 30 eggs per replicate

Reference substance (positive control):

no

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 46.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

number hatched

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 46.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 46.4 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: length and weight

Details on results:

- Hatching success: The fish started hatching on day 3 after start of exposure. Hatching was finished on day 8 for all treatments. No effects in treatment groups with respect to controls was observed (hatching success ≥ 90%).
- Post-hatch success and survival of fish: the survival of fish in the control group was 78.6%, whereas in all treatments up to the highest tested concentration, the post-hatch success was > 59.3%. The differences between treatment and control groups were not statistically significant.
- Weight and length of healthy fish at test end: no effects were observed in the length and weight of fishes in the treatment groups with respect to the control.
- Appearance: all surviving fish appeared healthy at the end of exposure, abnormalities were not observed.

Reported statistics and error estimates:

- Hatching success was evaluated with the Fisher's Exact binomial test, whereas post-hatch success (mortality), dry weight and length were evaluated with the Welch t-test. The normal distribution was checked with Shapiro-Wilk's test and the variance homogeneity with Levene's test.
The statistical software package ToxRat 2.10 Professional was used for the calculations.

Table 1: Cumulative numbers and percentage of hatched fish in the control and treatments (values of two pooled replicates per treatment; n=2)

Nominal concentration (µg CMI/L)	Introduced eggs	Hatched fish	Not hatched	% Not hatched
Control	60	56	4	6.7
1.13	60	58	2	3.3
3.59	60	60	0	0.0
11.3	60	58	2	3.3
35.9	60	55	5	8.3
113	60	59	1	1.7

Table 2: Post-hatch success (survival of hatched fish) assessed at test end in the control and treatments (values of two pooled replicates per treatment; n=2)

Nominal concentration (µg CMI/L)	Hatched fish (n)	Surviving fish (n)	Post-hatch mortality (% of hatched fish)	Post-hatch success (% of hatched fish)
Control	56	44	21.4	78.6
1.13	58	48	17.2	82.8
3.59	60	44	26.7	73.3
11.3	58	41	29.3	70.7
35.9	55	34	38.2	61.8
113	59	35	40.7	59.3

Table 3: Per-treatment individual mean dry weight in mg at test end (values of two pooled replicates per treatment; n=2)

Nominal concentration (µg CMI/L)	Mean	Minimum	Maximum
Control	7.98	7.56	8.40
1.13	5.88	5.76	5.99
3.59	7.91	7.38	8.45
11.3	7.49	7.47	7.51
35.9	5.66	5.36	5.97
113	6.54	6.34	6.74

Table 4: Per-treatment mean lengths in mm at test end (values of two pooled replicates per treatment; n=2)

Nominal concentration (µg CMI/L)	Mean	Minimum	Maximum
Control	14.42	13.76	15.08
1.13	13.63	13.27	13.98
3.59	14.39	14.08	14.70
11.3	14.46	13.92	15.00
35.9	13.68	13.39	13.98
113	14.33	14.26	14.39

Description of key information

The results of the key study indicated that chronic exposure of Fathead minnow to CMIT/MIT resulted in a 38d NOEC of 0.02mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.02 mg/L