Climbazole

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Remarks:

Integrated with the Fish Sexual Development Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-June to 2021-September

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

yes

Remarks:

Due to animal wellfare reasons a OECD 234 was designed such that the endpoints required by an OECD 210 could be evaluated

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

LC-MS/MS

Details on sampling:

Samples (8 mL) of test media from the control and each test concentration were taken for analysis weekly throughout the duration of the test. A single replicate was sampled on each occasion with alternate replicates sampled each time with the exception of Day 0 (start of exposure) and the end of the test when each replicate was sampled.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solutions and dilution water were pumped by means of a peristaltic pump through an in-line mixer. The media were then split into the replicate test vessels.
- Controls: dilution water without test item
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Japanese medaka
- Source: in-house laboratory breeded

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: The eggs were collected from the breeding groups and gently rolled to remove filaments.

POST-HATCH FEEDING
- Start date: 13th day
- Type/source of feed: Tetramin® flake food and/or live brine shrimp (Artemia) nauplii, (ca 24 or 48 hours old), depending on the size of the fish.
- Frequency of feeding: 1 to 3 times per day

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Hardness:

In Control: 40-83 mg/L as CaCO3;
In treatments: 52-87 mg/L as CaCO3

Test temperature:

25 ± 2ºC

pH:

see details on test conditions

Dissolved oxygen:

> 60% air saturation value

Salinity:

see details on test conditions

Conductivity:

see details on test conditions

Nominal and measured concentrations:

Nominal Concentrations: 0.0050, 0.016, 0.050, 0.16 and 0.50 mg/L.
Mean measured concentrations: 0.0053, 0.017, 0.052, 0.15 and 0.48 mg/L.

Details on test conditions:

TEST SYSTEM
- Emybro cups: glass scintillation vials
- Test vessel: constructed glass aquaria
- Type of flow-through: peristaltic pumpe
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:laboratory treated mains supply.The water was pumped to the laboratory through an activated carbon filter.
- Particulate matter: no (activated carbon filtered)
- Pesticides: analyzed but no significant findings
- Chlorine: 17 mg/L
- Ca/ Mg ratio: 6.7
- Alkalinity: 27.6 mg/L (as CaCO3)

OTHER TEST CONDITIONS
- Adjustment of pH: 7.6
- Photoperiod: 16 hour light:8-hour dark
- Light intensity: 588 to 838 Lux

RANGE-FINDING STUDY
- Test concentrations: 0-0.005-0.05-0.50-5.0 mg/L
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- Start post-hatch phase: 13 days following egg addition
- No. of hatched eggs (alevins)/treatment released to the test chamber: 30

Reference substance (positive control):

no

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

> 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

0.014 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

and weight (most sensitive endpoint)

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

adult mortality

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: no
- Incidents in the course of the test which might have influenced the results: no

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8.

Hatching Success: 

Nominal concentration (mg/L)	Hatching success (%)
	Mean	SD
Control	97	2.9
0.0050	96	3.4
0.016	99	1.7
0.050	94	5.1
0.16	99	1.7
0.50	99	1.5

30-day Post-hatch survial:

Nominal concentration (mg/L)	Post-hatch survival Day 30 (%)
	Mean	SD
Control	97	2.9
0.0050	92	1.5
0.016	96	3.4
0.050	51	10.0
0.16	0	0
0.50	0	0

30-Day Post-hatch Total Length and Wet Weight:

Nominal Concentration (mg/L)	Total length (mm)		Wet weight (mg)
	Mean	SD	Mean	SD
Control	15.1	2.1	38.8	13.8
0.0050	15.7	2.3	44.6	14.3
0.016	14.8	2.1	36.4	13.2
0.050	12.9	2.0	24.2	11.9

Validity criteria fulfilled:

yes

Conclusions:

Based on the most sensitive endpoint (length and weight), the EC10 of the effect of the test item to the early life stage of fish (Oryzias latipes) was determined to be 0.014 mg/L over 30-day post hatch phase.

Executive summary:

The long-term toxicity testing on fish with the substance was evaluated according to OECD TG 210 “Fish, Early Life Stage Toxicity Test (adopted July 2013)”. Due to animal wellfare reasons, this fish test was part of an OECD 234 "Fish Sexual Development Test" (see IUCLID Section 6.1.8). The OECD 234 was desinged such that the endpoints required by the OECD 210 could be evaluated. The tests were performed under GLP with the Japanse medaka (Oryzias latipes) using a flow-through system. Based on the results from a range-finding test, the definitive test was conducted at nominal test concentrations of 0.0050, 0.016, 0.050, 0.16 and 0.50 mg/L. A control group was also included. Four replicate vessels were used for the control and each test concentration. The concentrations were verified via a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) system. The measured concentrations were remained within the 80% to 120% of nominal concentrations during the test. Based on the most sensitive endpoint (length and weight), the EC10 of the effect of the test item to the early life stage of fish was determined to be 0.014 mg/L over 30-day post hatch phase.

Description of key information

The long-term toxicity testing on fish with the substance was evaluated according to OECD TG 210 “Fish, Early Life Stage Toxicity Test (adopted July 2013)”. Due to animal wellfare reasons, this fish test was part of an OECD 234 "Fish Sexual Development Test" (see IUCLID Section 6.1.8). The OECD 234 was desinged such that the endpoints required by the OECD 210 could be evaluated. The tests were performed under GLP with the Japanse medaka (Oryzias latipes) using a flow-through system. Based on the results from a range-finding test, the definitive test was conducted at nominal test concentrations of 0.0050, 0.016, 0.050, 0.16 and 0.50 mg/L. A control group was also included. Four replicate vessels were used for the control and each test concentration. The concentrations were verified via a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) system. The measured concentrations were remained within the 80% to 120% of nominal concentrations during the test. Based on the most sensitive endpoint (length and weight), the EC10 of the effect of the test item to the early life stage of fish was determined to be 0.014 mg/L over 30-day post hatch phase.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

0.014 mg/L