p-methoxybenzyl acetate

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:

2016-04-25 to 2016-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and Test concentrations (nominal): 1.88 - 3.75 - 7.50 - 15.0 - 30.0 mg/L (factor 2)
- Sampling method: The test concentrations and the control were analytically verified from freshly prepared media after 0, 24, 48 and 72 h and from corresponding aged test media after 24, 48, 72 and 96 h.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

- Method:Appropriate volumes of the test item were pipetted directly into the test vessels filled with appropriate volumes of the dilution water. The test media were mixed with an ultraturrax (1 minute, 12000 rpm). Thereafter, the test vessels were covered with a floating lid. This procedure was repeated for each renewal day.PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):The test item was clearly dissolved throughout exposure.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish, Gnathostoma, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Length at study initiation: mean 2.5 cm
- Weight at study initiation: mean 0.13 g

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: Holding was performed at the test facility at 23 ± 2 °C and diffuse light (0.1 -10 µmol photons * m*exp-2 * s*exp-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value.
- Type and amount of food during acclimation: Sera Vipan; Sera GmbH, 52518 Heinsberg, Germany; The amount of food was 4% of the fish body weight per feeding day.
- Feeding frequency during acclimation: Food was provided 3 times per week.
- Health during acclimation: No mortality was observed.

FEEDING DURING TEST
Fish were not fed during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

64 mg CaCO3/L

Test temperature:

22.8-24.1

pH:

7.02-7.63

Dissolved oxygen:

99-100% of saturation concentration

Salinity:

Not applicable

Conductivity:

172 pS/cm

Nominal and measured concentrations:

1.88, 3.75, 7.50, 15.0, 30.0 mg/L (nominal)
1.08, 2.77, 6.06, 13.9, 28.4 mg/L (geometric mean measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 13 L (dimensions: 25.5/18/28 cm)
- Type: closed, floating lid
- Material, size, headspace, fill volume: glass, 13 L, no headspace, 10 L
- Aeration: No aeration was provided.
- Renewal rate of test solution: Medium was renewed every 24 h.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: Fish density in the test vessels was 0.091 g fish per litre test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing.
- Chlorine: The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine.
- Alkalinity: 0.60 mmol/L
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all test vessels at the beginning and at the end of the exposure and every 24 hours from fresh and old test media. Total hardness of the water was determined at the beginning out of the control. During the test the water temperature was recorded once per hour with a data logger. The light intensity on the surface of the test aquaria was measured at test start.
- Acidity: 0.10 mmol/L

OTHER TEST CONDITIONS
- Photoperiod: A daily 16 hours photoperiod was maintained throughout exposure which corresponded to the natural daylight hours.
- Light intensity: 7 - 750 Lux

EFFECT PARAMETERS MEASURED:
Observations were made after 3, 24, 48, 72 and 96 h. Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). For measurements of size and weight the control fish were used at the end of the exposure.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

13.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % Confidence Interval: 6.06-13.9

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

6.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

28.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Animals treated with 28.4 mg/L showed hyperventilation and staggering after 3 h exposure. 5 animals treated with 13.9 mg/L showed missing escape reflex after 24 h exposure. After 48 h two animals showed staggering and 1 animal showed side position. After 72 and 96 h 1 animal showed staggering.
- Observations on body length and weight: Body weight and length averages were determined before the experiment start as 0.13 g and 2.5 cm. No observation was made during or after the exposure.
- Other biological observations: Mortality was observed for all animals treated with 28.4 mg/L after 24 h exposure. Treatment with 13.9 mg/L resulted in the death of 2 animals each after 24, 48 and 72 h of exposure. No mortality was observed for the other concentrations.
- Mortality of control: No mortality was observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
- Effect concentrations exceeding solubility of substance in test medium: No.

Reported statistics and error estimates:

After 24 to 96 hours only one partial response occurred. Therefore, the LC10 and LC20 values were given as a range and the LC50-values were calculated by sigmoidal regression. The concentrations enclosed to the LC50-values after 24, 48, 72 and 96 hours were chosen as lower and upper confidence limits. The LC0- and LC100-value were determined directly from the test results.
The geometric mean measured test item concentrations after 96 hours of exposure were used for all corresponding calculations. The chosen methods for determination of LCx-values were in accordance with the OECD series on testing and assessment No. 54 (2006).

Sublethal observations / clinical signs:

Measured Concentrations and Percent of the Nominal Concentrations of the Test Item

Sampling date	2016-05-30 0 h (Fresh medium)		2016-05-31 24 h (Old medium)		2016-05-31 24 h (Fresh medium)		2016-06-01 48 h (Old medium)		2016-06-01 48 h (Fresh medium)		2016-06-02 72 h (Old medium)		2016-06-02 72 h (Fresh medium)		2016-06-03 96 h (Old medium)
Nominal test item concentration [mg/L]	Test substance
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Geometric Mean measured test item conc. [mg/L]
30	30.5	102	26.4	88	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	28.4
15	15.1	101	12.2	81	15.7	104	13	86	15.7	105	11.6	77	15.6	104	13.3	89	13.9
7.5	6.79	91	5.76	77	6.83	91	5.61	75	7.1	95	4.52	60	6.58	88	5.78	77	6.06
3.75	3.13	83	2.51	67	3.16	84	2.61	70	3.22	86	2.26	60	2.92	78	2.51	67	2.77
1.88	1.06 (1)	57	0.794	42	1.34	71	0.98	52	1.32	70	0.864	46	1.31	70	1.11	59	1.08
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = Measured concentration of the test item, dilution factor taken into account

% = Percent of nominal concentration of the test item

n.d. = not determined, due to 100% mortality after 24h

LOQ = Limit of quantification (0.13 mg/L test item)

(1) = reanalysed on 2016-05-31; mean value of 3 replicates

 

Observations in the Definitive Test (n = 7)

Geometric Mean measured test item conc. [mg/L]	Observation *)	Number of fish effected after different exposure periods [h]
		3	24	48	72	96
28.4	(E)	-	7	---	---	---
	(4.1)	7	-	---	---	---
	(2.3)	7	-	---	---	---
13.9	(E)	-	2	2	2	---
	(2.5)	-	5	-	---	---
	(2.3)	-	-	2	1	1
	(2.2)	-	-	1	---	---
	(1)	7	-	-	---	---
6.06	(1)	7	7	7	7	7
2.77	(1)	7	7	7	7	7
1.08	(1)	7	7	7	7	7
Control	(1)	7	7	7	7	7

*) The numbers in brackets correspond to the following observations:

(1) = Normal behaviour

--- = No observations due to 100 % mortality

(2.2) = Side position

(2.3) = Staggering

(2.5) = Missing escape reflex

(4.1) = Hyperventilation

(E) = Exitus lethalis

 

Cumulative Mortality [%] in the Definitive Test (n = 7)

Geometric Mean measured test item conc. [mg/L]	Cumulative Mortality [%] after different exposure periods [hours]
	3	24	48	72	96
28.4	0	100	100	100	100
13.9	0	29	57	86	86
6.08	0	0	0	0	0
2.77	0	0	0	0	0
1.08	0	0	0	0	0
Control	0	0	0	0	0

 

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 for fish was determined to be 13.1 mg/L.

Executive summary:

The acute toxicity of the test substance to Zebrafish was determined according to OECD 203 (1992) and under GLP conditions. This study encompassed one treatment group for the dose rates of the test item and one replicate for the control. Each group contained 7 individuals. The acute toxicity to unfed juvenile Zebrafish (Danio rerio) was determined in semi-static, 96-hour test under 24 hours renewal conditions. The test concentrations and the control were analytically verified via HPLC-DAD from freshly prepared media and from corresponding aged test media. The nominal test concentrations were 0 (control), 1.88, 3.75, 7.50, 15.0 and 30.0 mg/L. The measured concentrations of the test substance based on the mean values were 1.08, 2.77, 6.06, 13.9 and 28.4mg/L. The measured concentrations in freshly prepared test media were in the range of 57 to 105 % of the nominal values. In corresponding 24 h aged test media, the measured concentrations were between 42 to 89 % of the nominal values. An additional peak was found in the chromatograms of 24 hours old solutions. It may be caused by a hydrolysis product of test item. All effect values are based on the geometric mean measured concentrations of the test item. The 96 hour LC50 was determined to be 13.1 mg/L.

Description of key information

The 96 hour LC50 for fish was determined to be 13.1 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

13.1 mg/L

Additional information

The acute toxicity of the test substance to Zebrafish was determined according to OECD 203 (1992) and under GLP conditions. This study encompassed one treatment group for the dose rates of the test item and one replicate for the control. Each group contained 7 individuals. The acute toxicity to unfed juvenile Zebrafish (Danio rerio) was determined in semi-static, 96-hour test under 24 hours renewal conditions. The test concentrations and the control were analytically verified via HPLC-DAD from freshly prepared media and from corresponding aged test media. The nominal test concentrations were 0 (control), 1.88, 3.75, 7.50, 15.0 and30.0 mg/L. The measured concentrations of the test substance based on the mean values were 1.08, 2.77, 6.06, 13.9 and 28.4mg/L. The measured concentrations in freshly prepared test media were in the range of 57 to 105 % of the nominal values. In corresponding 24 h aged test media, the measured concentrations were between 42 to 89 % of the nominal values. An additional peak was found in the chromatograms of 24 hours old solutions. It may be caused by a hydrolysis product of test item. All effect values are based on the geometric mean measured concentrations of the test item. The 96 hour LC50 was determined to be 13.1 mg/L.