Benzyl butyrate

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed according to the guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method: The stock solution prepared as 2g /4 liter, with the concentration of 500 mg/L and was kept for 4 hours stirring. After the completion of stirring, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 122.66 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, respectively.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra Fish
- Strain: Danio rerio
- Length at study initiation (length definition, mean, range and SD): average 3.65 cm
- Weight at study initiation (mean and range, SD): average 0.332 g
-Housing:The fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration.
-Duration of housing: 15 days
-Photoperiod: 12 hours light and 12 hours darkness
-Diet : Standard brand feed, once daily
-Acclimatization: seven days
-feeding frequency: Once in a day

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

141.4 mg of CaCO3

Test temperature:

23°C

pH:

7.07

Dissolved oxygen:

7 to 7.2

Nominal and measured concentrations:

Nominal concentrations: 6.25 mg/L, 12.5 mg/L, 25mg/L, 50mg/L, 100 mg/L, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Material, size, headspace, fill volume: 5 liters of PP aquarium filled with 2 liter of water
- Aeration: Aeration in test vessels was provided 1 day before the start of experiment
- No. of organisms per vessel: 8 fishes

OTHER TEST CONDITIONS
- Photoperiod: 12 hours light and 12 hours darkness

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Other details not known

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: No effects were observed

Sublethal observations / clinical signs:

Table 1: Mortality and symptoms:

NominalConcentration (mg/l)	Number of fish		Mortality (cumulated) after
			3 h		6 h		24h			48h		72h		96h
			V1	V2	V1	V2	V1	V2		V1	V2	V1	V2	V1	V2
0 (control)	4	4	-	-	-	-	-		-	-	-	-	-	-	-
6.25	4	4	-	-	-	-	-		-	-	-	-	-	-	-
12.5	4	4	-	-	3	1	3		1	3	1	3	1	3	1
25	4	4	-	-	4	4	4		4	4	4	4	4	4	4
50	4	4	4	4	4	4	4		4	4	4	4	4	4	4
100	4	4	4	4	4	4	4		4	4	4	4	4	4	4

Table 2: Visible Symptoms:

NominalConcentration (mg/l)	Number of fish		Symptoms after
			3 h		6 h		24h			48h		72h		96h
			V1	V2	V1	V2	V1	V2		V1	V2	V1	V2	V1	V2
0 (control)	4	4	NS4	NS4	NS4	NS4	NS4		NS4	NS4	NS4	NS4	NS4	NS4	NS4
6.25	4	4	NS4	NS4	NS4	NS4	NS4		NS4	NS4	NS4	NS4	NS4	NS4	NS4
12.5	4	4	NS4	NS4	C3NS1	C2NS2	NS1		NS3	NS1	NS3	NS1	NS3	NS1	NS3
25	4	4	NS4	C2NS2	-	-	-		-	-	-	-	-	-	-
50	4	4	-	-	-	-	-		-	-	-	-	-	-	-
100	4	4	-	-	-	-	-		-	-	-	-	-	-	-

Explanation of symptoms:

A:apathy

N:Narcotic-like state

C:Swimming near water surface

M: Slow movements compared to control

NS:No abnormal symptoms 

S:Surfacing behaviour

L:Loss of equilibrium

SM:Shimming movement

H1:Hyperactivity

H2:Hypo activity

B:Bending behavior

F:Fin rot

IS:Invert Swimming

SB:Swimming behaviour

RF:Respiratory Function

P:Pigmentation

Table 3: pH and oxygen content:

NominalConcentration (mg/l)	Number of fish		pH readings after
			24h			48h		72h		96h
			V1	V2		V1	V2	V1	V2	V1	V2
0 (control)	4	4	8.11		8.12	7.89	7.84	7.84	7.89	8.08	8.10
6.25	4	4	8.23		8.24	7.71	7.74	7.64	7.55	8.17	8.21
12.5	4	4	8.14		8.18	7.92	7.97	7.93	7.95	8.22	8.26
25	4	4	-		-	-	-	-	-	-	-
50	4	4	-		-	-	-	-	-	-	-
100	4	4	-		-	-	-	-	-	-	-

NominalConcentration (mg/l)	Number of fish		Oxygen content (mg/L) after
			24h			48h		72h		96h
			V1	V2		V1	V2	V1	V2	V1	V2
0 (control)	4	4	7.0		7.1	7.0	7.1	7.1	7.1	7.0	7.0
6.25	4	4	7.1		7.1	7.1	7.1	7.0	7.0	7.0	7.0
12.5	4	4	7.2		7.1	7.1	7.0	7.0	7.0	7.0	7.0
25	4	4	-		-	-	-	-	-	-	-
50	4	4	-		-	-	-	-	-	-	-
100	4	4	-		-	-	-	-	-	-	-

Table 4: Test temperature

NominalConcentration (mg/l)	Number of fish		Temperature (°C) after
			24h			48h		72h		96h
			V1	V2		V1	V2	V1	V2	V1	V2
0 (control)	4	4	23		23	23	23	23	23	23	23
6.25	4	4	23		23	23	23	23	23	23	23
12.5	4	4	23		23	23	23	23	23	23	23
25	4	4	-		-	-	-	-	-	-	-
50	4	4	-		-	-	-	-	-	-	-
100	4	4	-		-	-	-	-	-	-	-

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra Fish Danio rerio was determine to be 12.5 mg/l.

Executive summary:

The acute study was designed to access the toxic effects of the test compound on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. In house solubility was observed to be 281.985 mg/L. Thus the stock solution prepared as 2g /4 liter, with the concentration of 500 mg/L and was kept for 4 hours stirring. After the completion of stirring, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 122.66 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra Fish Danio rerio was determine to be 12.5 mg/l. Thus on the basis of LC50 value, test chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria, but as the chemical was readily biodegradable in water, hence chemical can be consider to be nontoxic and not classified as per the CLP classification criteria.

Description of key information

The acute study was designed to access the toxic effects of the test compound on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. In house solubility was observed to be 281.985 mg/L. Thus the stock solution prepared as 2g /4 liter, with the concentration of 500 mg/L and was kept for 4 hours stirring. After the completion of stirring, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 122.66 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra Fish Danio rerio was determined to be 12.5 mg/l. Thus on the basis of LC50 value, test chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria, but as the chemical was readily biodegradable in water, hence chemical can be consider to be nontoxic and not classified as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

12.5 mg/L

Additional information

Following different studies includes experimental study for the test chemical and read-across analogues which is extracted by using mechanistic approach and functionally and structurally similar to the target chemical to observe the short-term toxicity of test chemical to fishes.

 

The acute study was designed to access the toxic effects of the test compound on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. In house solubility was observed to be 281.985 mg/L. Thus the stock solution prepared as 2g /4 liter, with the concentration of 500 mg/L and was kept for 4 hours stirring. After the completion of stirring, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 122.66 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra Fish Danio rerio was determined to be 12.5 mg/l. Thus on the basis of LC50 value, test chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria, but as the chemical was readily biodegradable in water, hence chemical can be consider to be nontoxic and not classified as per the CLP classification criteria.

 

Above study further supported by the second study from experimental source. This study was designed to access the toxic effects of the test chemical on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. The stock solution was prepared by dissolving 1ml of the test substance in 1 liter of potable water (passed through reverse osmosis system) with 24 hrs of continuous stirring. From this stock solution, further test concentration was prepared for achieving test concentrations of 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L & 100 mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. The median lethal concentration (LC50) value of test chemical on Danio rerio in a 96 hours study on the basis of mortality effect was determined to be >12.5 mg/L. As 100 % mortality was observed at the concentration of 25 mg/l, thus on that basis, chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria, but as the chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

 

Similar short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. The study was performed following the OECD Guideline 203 (Fish, Acute Toxicity Test) under flow through conditions. Oryzias latipes (Japanese Medaka) of 28 -43 days old and 18 -71 mg weight obtained from the Environmental Research Laboratory-Duluth (ERL-D) culture unit was used as a test organism. Test fishes were nurtured in tank at 25°C and fed with live Biomaine brand brine shrimp. Before 24 hr or during the study, test organism was not fed. 5 different concentrations of test chemical along with the control were taken for the study. Stock solutions of test chemical was prepared by dissolving the test chemical in Lake Superior water, using a high speed stirrer. Stock solutions were then transferred to a glass stock bottle inside the vented diluter enclosure using Teflon tubing and air pressure. During each test, a predetermined volume (ml/min) of stock solution was continuously pumped from the stock bottle into the mixing cell of the diluter system. Test chemical concentrations were verified analytically and analysis was carried out bya Hewlett Packard 5730A gas chromatograph (GC) equipped with a flame ionization detector (FID) linked to an HP 3350 lab automation system. All test analyses were accomplished using direct aqueous injection. GC column consists of a wall-coated open tubular silica column, 0.53 mm I.D. x 15 cm, with a 2.5 µ phase of bonded polyethlene glycol at isothermal oven temperature of 85, 120 and 110°C, respectively. Total 20 fishes/conc (10 organisms/replicate) were exposed to test chemical in a2.0 lit glass aquaria tank.2.0 l glass aquaria tank has a dimension of 18.5 X 14.0 X 13.0 cm deep. It has a 8.6 cm standpipe which resulted in a total volume of 2.0 lit. Continuous-flow mini diluter exposure system with vented enclosures was used for the study. Flow rate during the study was 25 ml/min with 90% replacement times of 2.8 hr. Lake superior water was used.It was filtered before use through sand, a 50-micron filter; a 5-micron filter; and then exposed to ultraviolet light before heating to the test temperature of 25±1°C.The test vessels were placed in a room at a temperature of 25±1°C, pH 7.88 ± 0.18 (7.31 to 8.85), dissolve oxygen (D. O) 6.8 ± 0.7 (5.0 to 8.5), hardness of water 45.8 (38.0 to 52.0) mg/l as CaCO3, alkalinity 45.9 (35.0 to 58.5 mg/l) as CaCO3 and under a 16 hr photoperiod with a light intensity of 12 to 25 lumens provided by fluorescent lamps for 96 hrs. All experiments were performed in replicate.95% confidence intervals were calculated using the binomial tests. Dissolved oxygen (D.O.) was measured by a dissolved oxygen meter. pH was determined on one set of replicate tanks atleast once and often twice during the test. On the other hand, hardness and alkalinity determinations were done at a minimum on a control, one intermediate and one at high test concentration tank; it was carried out once or twice during the study. Mortality was noted after an exposure period of 96 hrs. No mortalities were observed in the control vessel, the dissolved oxygen concentration was evaluated to be ≥ 60% (i.e, reported as 82.3%) of the air saturation value in test vessels throughout the study period and analytical monitoring of test concentrations has been carried out, thus fulfilling the validity criteria of the study. As the test concentrations were maintained within ±20% of the initial measured concentrations throughout the study, all results will be reported in nominal concentrations. On the basis of the effect on mortality of the test organism Oryzias latipes (Japanese Medaka), the 96 hr LC50 was determined to be 4.0 mg/l (95% C. I. = 3.48 to 4.60 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to fish and hence, considered to be classified in ‘aquatic chronic category 2’ as per the CLP classification criteria. But as the test chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Thus, on the basis of above all studies and effects observation, it is concluded that the test chemical was nontoxic and not classified as per the CLP classification criteria.