Myrac Aldehyde

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:

From 19-01-11 to 19-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Not relevant.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 2018-11-15

Analytical monitoring:

yes

Details on sampling:

- Concentrations: a limit test was performed at the nominal loading rate of 0.50 mg.L-1 in order to achieve an average exposure concentration close to 0.43 mg.L-1 (based on the results of a stability pre-test performed under experimental conditions and without fish).
- Sampling method: Single samples for analysis were taken from the control and all test solutions at the start (t=0h) and every 24 hours thereafter in old and new solutions.

Vehicle:

no

Details on test solutions:

The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h, t=24h, t=48h and t=72h) were prepared under closed conditions and by slow-stirring.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of the mixing vessels was approximately 5 L. A magnetic stirring bar was placed in the vessels and 5.2 L to 5.7 L of test water (depending on the brim capacity of the bottle) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. The mixing vessels were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring (except for preparation of fresh solutions at t=0h where mixing was carried out throughout the weekend) in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed after filling and introduction of fish with a minimum of headspace. The test solutions in test vessels were observed to be clear and colourless. The test was carried out without adjustment of the pH.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species Zebrafish (Danio rerio)
Origin Elevage de la Grande Rivière - B. et C. Romano - La Fond Garel – 69490 SAINT FORGEUX
Sex Male and female
Reason for selection The system has been selected as an internationally accepted species.
Feeding Daily with flaked food or live food (such as brine shrimp nauplii).
Validity of batch In the batch of fish which was used for the test, no mortality during the seven days prior to the start of the test was observed. Moreover, the average length and weight of the fish used during the final test were 2.65 cm and 0.241 g, respectively. Hence, this batch was considered valid.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

Between 10 and 250 mg.L-1 (as CacO3).

Test temperature:

23°C ± 2°C

pH:

Between 6.0-8.5

Dissolved oxygen:

Between 76.0% and 97.6% of the air saturation value throughout the test.

Salinity:

No data.

Conductivity:

< 10 µS.cm-1

Nominal and measured concentrations:

Nominal loading rate (limit test) : 0.50 mg.L-1.

Details on test conditions:

PREPARATION OF STOCK AND TEST SOLUTIONS
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h, t=24h, t=48h and t=72h) were prepared under closed conditions and by slow-stirring.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of the mixing vessels was approximately 5 L. A magnetic stirring bar was placed in the vessels and 5.2 L to 5.7 L of test water (depending on the brim capacity of the bottle) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. The mixing vessels were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring (except for preparation of fresh solutions at t=0h where mixing was carried out throughout the weekend) in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed after filling and introduction of fish with a minimum of headspace. The test solutions in test vessels were observed to be clear and colourless. The test was carried out without adjustment of the pH.

TEST PROCEDURE AND CONDITIONS
Test duration 96 hours
Test type A semi-static test was performed, with daily renewal of the test solutions and the control in closed test vessels, in order to maintain the dissolved oxygen concentration higher than 60 % of the air-saturation value throughout the test, and to prevent the dissipation of the test item from test solutions.
Test vessels All-glass aquaria, of 3.5 L capacity filled with minimum headspace (approximately 1 cm) and closed by placing a glass plate on the aquarium and sealing with Vaseline.
Each test vessel was uniquely identified with study code, date of experimentation and treatment group.
Number of fish/loading 7 fish per vessel filled with minimum headspace, with a fish loading not exceeding 1.0 g.L-1
Light regime 16h light : 8 h dark
Aeration No aeration of the test solutions occurred throughout the test.
Feeding No feeding from 24 hours prior to the test and during the total test period.
Introduction of fish Fish were introduced into the test medium immediately after filling of test vessels with test solutions. Ten fish of the batch used for the final test were measured at the end of the test (average length: 2.85 cm).
Mortality and other effects Fish were inspected at 3, 6, 24, 48, 72 and 96 hours following the start of exposure. Dead fish recorded were removed when observed.
Euthanasia At the end of the test, the surviving fish were rapidly euthanised by exposing them to benzocaine at a concentration of 200 mg.L-1.
Holding and test water Reconstituted water, as prescribed by the OECD Guideline 203.
Stock solutions: a) CaCl2.2H2O 11.76 g.L-1
b) MgSO4.7H2O 4.93 g.L-1
c) NaHCO3 2.59 g.L-1
d) KCl 0.23 g.L-1
An aliquot of each solution (a to d) was added to each litre (final volume) of deionised water (conductivity < 10 µS.cm-1).
The pH of this solution was in the range of 6 to 8.5 and the water hardness was between 10 and 250 mg.L-1 (as CaCO3).
Measurements of pH and dissolved O2 Measured daily in all vessels.
Measurements of Temperature of medium: Measured daily in all vessels with fish; temperature was measured continuously in the control vessel and in the limit test concentration.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: loading rate

Basis for effect:

mortality (fish)

Details on results:

A limit test was performed at the nominal loading rate of 0.50 mg.L-1 in order to achieve an average exposure concentration (threshold concentration) close to 0.43 mg.L-1, according to the threshold approach for acute fish toxicity testing (OECD Guidance No. 126 (8)).
As explained above, the evaluation of the effects on D. rerio was based on the geometric means of the measured concentrations for the single-concentration: 0.23 mg.L-1. This average concentration was a little far from the target exposure concentration of 0.43 mg.L-1. Nevertheless, this difference was due as explained above to test item metabolization by fish, which dropped the geometric mean of the measured concentrations. However, regarding initial measured concentrations with fish and fresh/24h-old solutions in the abiotic condition, it can be assumed that fish were clearly exposed to the test item at an average exposure concentration very close to (and even higher than) 0.43 mg.L-1.
After 3, 6, 24, 48, 72 and 96 hours of exposure, no (sublethal or lethal) effect on the test animals was observed in the control and the limit test concentration. The absence of effect at the limit test concentration indicates that the fish is not the most sensitive group of test organism after short-term exposure and that, with at least 99% of confidence, the LC50 is greater than the threshold concentration, according to OECD Guidance No. 126.

Results with reference substance (positive control):

On August 2, 2018 (KP18-001; most recent test), the 24h-LC50 was 144.0 mg.L-1. Hence, the sensitivity of this batch of Danio rerio was in agreement with internal historical data.

Reported statistics and error estimates:

None.

Sublethal observations / clinical signs:

Water quality parameters and environmental conditions throughout the test

Test conditions remained within the ranges prescribed by the study plan ([O₂] > 60% of air saturation; temperature: 23°C ± 2°C, constant within 2°C).

The pH values measured in test vessels during the study were situated between 6.0 and 8.5 and were constant within one unit, except for the control where a gap of 1.21 units was recorded between the start and the end of the test. This deviation was considered not to affect the results of the test as no impact was observed on the controls throughout the duration of the test (i.e. no mortality or behavioural abnormalities).

pH-values during the final test

 

		Nominal concentration and corresponding measured concentration (geometric mean) (mg test item.L^-1)
		Control (0)	0.5 (0.23)	0.5Abiotic (0.57)
Start t=0h		7.24	7.45	7.54
t=24h	Old	6.67	7.22	7.63
	Fresh	7.17	7.40	7.69
t=48h	Old	7.81	7.56	7.81
	Fresh	7.88	7.83	7.89
t=72h	Old	6.92	7.51	7.97
	Fresh	7.70	7.76	7.98
End t=96h		7.36	7.32	7.57

Dissolved oxygen concentrations (mg.L^-1) and corresponding air saturation values (%) during the final test

 

		Nominal concentration and corresponding measured concentration (geometric mean) (mg test item.L^-1)
		Control (0)	0.5 (0.23)	0.5Abiotic (0.57)
Start t=0h		7.93 [88.0%]	7.69 [84.4%]	7.45 [82.7%]
t=24h	Old	6.64 [74.0%]	7.00 [78.0%]	7.90 [88.6%]
	Fresh	8.17 [89.5]	8.16 [89.8%]	8.15 [89.7%]
t=48h	Old	6.80 [76.0]	7.01 [79.3%]	8.37 [95.0%]
	Fresh	8.84 [97.6%]	8.75 [95.9%]	8.80 [97.5%]
t=72h	Old	6.94 [78.9]	6.92 [78.6%]	8.31 [94.3%]
	Fresh	8.31 [93.8%]	8.32 [94.1%]	8.27 [93.5%]
End t=96h		7.04 [79.4%]	6.78 [76.8%]	8.38 [95.3%]

 

 

Temperatures (°C) measured during the final test

 

		Nominal concentration and corresponding measured concentration (geometric mean) (mg test item.L^-1)
		Control (0)	0.5 (0.23)
Start t=0h		21.2	22.7
t=24h	Old	21.5 Min 21.2 Max 21.6	23.2 Min 22.7 Max 23.4
	Fresh	21.2	22.8
t=48h	Old	21.6 Min 21.2 Max 21.8	23.2 Min 22.8 Max 23.4
	Fresh	21.6	23.4
t=72h	Old	21.6 Min 21.4 Max 21.8	23.3 Min 23.3 Max 23.4
	Fresh	21.6	23.4
End t=96h		21.6 Min 21.4 Max 21.6	23.4 Min 23.2 Max 23.4

Min: lowest temperature recorded; Max: highest temperature recorded.

 

 

Incidence of mortality and total mortality during the final test.

 

	Nominal concentration and corresponding measured concentration (geometric mean) (mg test item.L^-1)
Nominal Cumulative mortality	Control (0) M     S	0.5 (0.23) M     S
t=3h	0      0	0      0
t=6h	0      0	0      0
t=24h	0      0	0      0
24h Total mortality (%)	0	0
t=48h	0      0	0      0
48h Total mortality (%)	0	0
t=72h	0      0	0      0
72h Total mortality (%)	0	0
t=96h	0      0	0      0
96h Total mortality (%)	0	0

M: Number of dead fish; S: Number of surviving fish but with sublethal effects. The initial number of fish in each test vessel was 7.

Analytical results

Samples taken from the control and the single-concentration (with and without fish) were analysed at the start (t=0h) and every 24 hours thereafter in old and new solutions in order to determine maintenance of actual concentrations of the test item.

Although every effort was made to maintain the concentrations of the test item (especially daily renewal of the test solutions and closed conditions with minimum headspace), the loss of the substance was greater than 20% of the initial (and nominal) concentration. Based on the log Kow of the test item it was suspected that these losses were due to the adsorption/accumulation of the test substance in fish. This assumption was reinforced by the fact that test item concentrations in abiotic treatments were relatively stable and within 80% of the initial concentrations throughout the duration of the test. Moreover, analytical results confirmed that in presence of fish the disappearance of test item in 24h-old solutions was correlated with the appearance of a new chromatographic peak, which strengthen the hypothesis of an uptake and metabolism of the test item by fish, with degradation product formation.Since the deviation of the exposure concentrations of the test item was greater than ± 20% of the initial concentrations in presence of fish, the results were expressed in terms of geometric means of the exposure concentrations for the present study.

Concentrations of the test item (mg.L^-1) in test water - Final test.

3-CYCLOHEXENE-1-CARBOXALDEHYDE MULTICONSTITUENT (99.50% of the test substance).

 

Nominal concentration (mg.L^-1)	Start (t=0h)		t=24hold		Relative loss to initial value (t=0h - t=24hold) (%)	t=24hfresh		t=48hold		Relative loss to initial value (t=24hfresh- t=48hold) (%)
	Meas. Conc(mg.L^-1)	% nominal	Meas. Conc(mg.L^-1)	Meas. Conc(mg.L^-1)		Meas. Conc(mg.L^-1)	% nominal	Meas. Conc(mg.L^-1)	% nominal
Control	Abs.	N.A.	Abs.	Abs.	N.A.	Abs.	N.A.	Abs.	Abs.	N.A.
0.50	0.60	120	0.21	42	66	0.57	114	0.15	30	73
0.50Abiotic	0.76	152	0.62	124	18	0.57	114	0.52	104	8

 

Nominal concentration (mg.L^-1)

t=48hfresh

t=72hold

Relative loss to initial value (t=48hfresh- t=72hold) (%)

t=72hfresh

End t=96h

Relative loss to initial value (t=72hfresh- t=96) (%)

Geometric mean measured concentrations

Meas. Conc(mg.L^-1)

% nominal

Meas. Conc(mg.L^-1)

Meas. Conc(mg.L^-1)

Meas. Conc(mg.L^-1)

% nominal

Meas. Conc(mg.L^-1)

% nominal

mg.L^-1

% nominal

Control

Abs.

N.A.

Abs.

Abs.

N.A.

Abs.

N.A.

Abs.

Abs.

N.A.

N.A.

N.A.

0.50

0.48

96

Pres.

N.A.

N.A.

0.52

N.A.

Pres.

N.A.

N.A.

0.23

46

0.50Abiotic

0.59

118

0.50

100

16

0.55

110

0.51

102

7

0.57

114

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

Pres.= Presence: concentrations below the LOQ (0.11 mg.L^-1) but above the LOD (0.03 mg.L^-1). Where measured concentrations were below the LOQ (0.11 mg.L^-1) but above the LOD (0.03 mg.L^-1), such concentrations were considered to be the half of the quantification limit, according to the study plan (àLOQ/2 = 0.055 mg.L^-1).

Abs.= Absence: concentrations below the LOQ (0.11 mg.L^-1) and the LOD (0.03 mg.L^-1).

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of test item 3-CYCLOHEXENE-1-CARBOXALDEHYDE MULTICONSTITUENT to the zebrafish (Danio rerio) was investigated in a closed semi-static limit test. Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 96-hour LC50 value was determined to be higher than 0.23 mg.L-1.

Executive summary:

The purpose of this study was the assessment of the test item for its ability to generate acute toxic effects in Danio rerio (Zebrafish) during an exposure period of 96 hours in a semi-static test.

A study was performed to assess the acute toxicity of the test item 3-CYCLOHEXENE-1-CARBOXALDEHYDE MULTICONSTITUENTto the zebrafish Danio rerio. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test", referenced as Method C.1 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Based on the results of an acute toxicity test on D. magna (48h-EC₅₀= 0.43 mg.L^-1, measured concentration - OECD 202) and an algal toxicity test (72h-EC₅₀≥ 0.88 mg.L^-1, measured concentration - OECD 201), a limit test was performed at the nominal loading rate of 0.50 mg.L^-1 in order to achieve an average exposure concentration (threshold concentration) close to 0.43 mg.L^-1, according to the threshold approach for acute fish toxicity testing (OECD Guidance No. 126).The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 3, 6, 24, 48, 72 and 96 hours. Samples taken fromthe control and thesingle-concentration (with and without fish) were analysed at the start of the test and every 24 hours thereafter in old and new solutions.

Although every effort was made to maintain the concentrations of the test item (especially daily renewal of the test solutions and closed conditions with minimum headspace), the loss of the substance was greater than 20% of the initial (and nominal) concentration. Based on the log Kow of the test item it was suspected that these losses were due to the adsorption/accumulation of the test substance in fish. This assumption was reinforced by the fact that test item concentrations in abiotic treatments were relatively stable andwithin 80% of the initial concentrations throughout the duration of the test. Since the deviation of the exposure concentrations of the test item was greater than ± 20% of the initial concentrations in presence of fish, the evaluation of the effects on D. rerio was based onthe geometric means of the measured concentrations for the single-concentration: 0.23 mg.L^-1.After 3, 6, 24, 48, 72 and 96 hours of exposure, no (sublethal or lethal) effect on the test animals was observed in the control and the limit test concentration.

Test item 96h-LC50 = 0.23 mg.L-1

Description of key information

The toxic effect of test item 3-CYCLOHEXENE-1-CARBOXALDEHYDE MULTICONSTITUENT to the zebrafish (Danio rerio) was investigated in a closed semi-static limit test and the 96-hour LC50 value was determined to be higher than 0.23 mg.L-1 (geometric mean of exposure measured concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.23 mg/L