Butyl O-butyryllactate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2018 to 21 Jun 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures

Version / remarks:

OECD series on testing and assessment number 23, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Butyl Butyryl Lactate
Appearance: Colourless liquid
Purity/Composition: 99.77% (assumed 100% for testing)
Test item storage: At room temperature

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 3).
Frequency at t=0 h and t=48 h
Volume 4.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 4.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The batch of Butyl Butyryl Lactate tested was a colourless liquid with a purity of 99.77% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with a loading rate of 100 mg/L applying three days of gentle magnetic stirring to ensure maximum dissolution of the test item in test medium. The obtained mixture was allowed to settle for a period of 120-144 minutes. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20% , presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding:
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.


Culture medium: M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
The following salts and vitamins were added to freshly prepared test medium (see section 4.8.2.2) to reach the following concentrations:
Salts: H3BO3 0.73 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.092 mg/L
LiCl 0.078 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.039 mg/L
Na2MoO4.2H2O 0.016 mg/L
NaBr 0.0041 mg/L
CuCl2.2H2O 0.0043 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0033 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00059 mg/L
Na2EDTA.2H2O 0.64 mg/L
Na2SiO3.5H2O 7.6 mg/L
NaNO3 0.28 mg/L
KH2PO4 0.15 mg/L
K2HPO4 0.19 mg/L

Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

18-22 °C

pH:

6-9

Dissolved oxygen:

7.4-8.5 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal: Solutions containing 10, 18, 32, 56 and 100% of the SS (saturated solution), prepared at a loading rate of 100 mg/L.
Measured mean: 3.4, 7.3, 12, 23, 45 Average conc. (mg/L)

Details on test conditions:

The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and the undiluted SS prepared at a loading rate of 100 mg/L, in the limit test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to solutions containing 1.0 and 10% of the SS prepared at 100 mg/L, in the combined range-finding test.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass.
Test medium: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9.
Number of daphnids 20 per concentration
Loading 5 per vessel containing 50 mL of test solution.
Light A daily photoperiod of 16 hours.
Feeding No feeding
Aeration No aeration of the test solutions was applied.
Introduction of daphnids Within 25 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

Test medium without test item or other additives.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Combined Limit/Range-Finding Test:
Table 1 shows the responses recorded during the combined limit/range-finding test.

No immobility was observed in the control and at the two lowest test concentrations throughout the test. At the end of the test, 65% immobility was observed at the highest concentration.

Based on these results, samples taken from solutions containing 10 and 100% of the SS prepared at a loading rate of 100 mg/L were analysed. The concentrations measured at the start of the test were 6.7 and 70 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 83-84% of the initial concentration at the end of the test (see also Table 2 of the appended Analytical Report).

All test conditions were maintained within the limits prescribed by the study plan.

Final Test:
Measured Test Item Concentrations:
The results of analysis of the samples taken during the final test are described in Table 3 of the appended Analytical Report.

Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 4.4, 8.9, 13, 24 and 46 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at 100 mg/L, respectively. At the end of the test, the concentrations were at 60-97% relative to the initial concentrations.

Based on the obtained results, average exposure concentrations were calculated and used to determine the effect parameters (see Table 2).

Immobility:
Table 3 shows the responses recorded during the final test. No immobility was observed in the control and at the three lowest test concentrations throughout the exposure period. At the end of the test, 10 and 95% immobility were observed at average exposure concentrations of 23 and 45 mg/L, respectively.

The responses recorded in this test allowed for reliable determination of an EC50.

Determination of Effect Concentrations:
Table 4 shows the effect parameters based on average exposure concentrations, see also Appendix 1.
Table 4
Effect Parameters

Experimental Conditions:
The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 5. These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: 3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 20 and 22°C during the test, and complied with the requirements as laid down in the study plan (18 22°C, constant within ±1°C).

Results with reference substance (positive control):

See Appendix 2, "Reference Test", attached below.

Reported statistics and error estimates:

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Table 1
Number of Introduced Daphnids and Incidence of Immobility in the Combined Limit/Range-Finding Test

 

Time (h)	Replicate	Butyl Butyryl Lactate;%SS prep. at 100 mg/L
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	1
	B	0	0	0	1
	C	0			2
	D	0			0
	Total immobilised	0	0	0	4
	Effect %	0	0	0	20
48	A	0	0	0	1
	B	0	0	0	4^#
	C	0			4
	D	0			4
	Total immobilised	0	0	0	13
	Effect %	0	0	0	65

^#Microscopic observation revealed no test item attached to the daphnids.

Table 2
Average Exposure Concentration vs. Percentage of the Saturated Solution

 

Butyl Butyryl Lactate %SS prep. at 100 mg/L	Measured concentrations (mg/L)		Average conc. (mg/L)
	t=0h	t=48h
10	4.38	2.61	3.4
18	8.91	6.02	7.3
32	13.1	10.3	12
56	24.1	21.9	23
100	46.0	44.5	45

Table 3
Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)	Replicate	Butyl Butyryl Lactate; Average conc. (mg/L)
		Control	3.4	7.3	12	23	45
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	0	3 (2)
	B	0	0	0	0	0	4
	C	0	0	0	0 (1)	0	1 (1)
	D	0	0	0	0	0 (1)	3^#
	Total immobilised	0	0	0	0	0	11
	Effect %	0	0	0	0	0	55
48	A	0	0	0	0	0	5
	B	0	0	0	0	1	5
	C	0	0	0	0	0	5
	D	0	0	0	0	1	4
	Total immobilised	0	0	0	0	2	19
	Effect %	0	0	0	0	10	95

( ) number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

^#Microscopic observation revealed no test item attached to the daphnids.

Table 4
Effect Parameters

Parameter	Butyl Butyryl Lactate Average conc. (mg/L)	95%-confidence interval (mg/L)
24h-EC50	43	37 – 50
48h-EC50	31	27 – 36

Table 5
pH and Oxygen Concentrations (mg/L) During the Final Test

Butyl Butyryl Lactate Average exposure conc. (mg/L)	Start (t=0 h)		End (t=48 h)
	O2	pH	O2	pH
Control	8.2	8.2	8.2	8.2
3.4	8.3	8.1	8.2	8.2
7.3	8.3	8.1	8.4	8.1
12	8.4	8.1	8.2	8.0
23	8.5	8.0	8.2	7.9
45	8.5	8.0	7.4	7.8

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Butyl Butyryl Lactate was 31 mg/L based on average exposure concentrations (95% confidence interval between 27 and 36 mg/L).

Executive summary:

The objective of the study was to evaluate Butyl Butyryl Lactate for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC₅₀ at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Butyl Butyryl Lactate tested was a colourless liquid with a purity of 99.77% and not completely soluble in test medium at the loading rate initially prepared.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed. The concentrations measured at the start of the test were 4.4, 8.9, 13, 24 and 46 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at 100 mg/L, respectively. At the end of the test, the concentrations were at 60-97% relative to the initial concentrations.

Based on these results, average exposure concentrations were calculatedto be 3.4, 7.3, 12, 23 and 45 mg/L.

No immobility was observed in the control and at the three lowest test concentrations throughout the exposure period. At the end of the test, respectively 10 and 95% immobility was observed at average exposure concentrations of 23 and 45 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to Butyl Butyryl Lactate was 31 mg/L based on average exposure concentrations (95% confidence interval between 27 and 36 mg/L).

Description of key information

Data generated using generally accepted scientifically methods taken from GLP certified study report.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

31 mg/L

Additional information

The result does not trigger classification as the registered substance is readily biodegradable.