Butyl O-butyryllactate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

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:

Frequency: at t=0 h, t=24 h and t=72 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 with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 32% of the SS but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 4.0 mL were taken from all test solutions for possible analysis. If not already 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 2 hours. 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.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Any residual volumes were discarded.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Stock culture:
Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium:
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture:
Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

°C

pH:

8.1 ± 0.2

Dissolved oxygen:

not reported

Salinity:

Not applicable

Conductivity:

not reported

Nominal and measured concentrations:

Nominal: Butyl Butyryl Lactate %SS prep. at 100 mg/L: 10, 18, 32, 32*, 56, 100
*Without algae
Measured mean: TWA conc. (mg/L): 2.0, 3.7, 6.6, 5.4, 11, 22

Details on test conditions:

The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a saturated solution 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:
• Three replicates per concentration were exposed to 1.0 and 10% of the SS prepared at a 100 mg/L, in the combined range-finding test.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

Test vessels 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
Test Medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L

Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity within the range of 89 to 90 µE.m-2.s-1.
Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Final Test:

Control: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

Measurements and Recordings:
pH: At the beginning and at the end of the test, for all test concentrations and the control
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells : At the end of the final test, microscopic observations were performed on the solution containing 56% of the SS and the control to observe for any abnormal appearance of the algae.

Recording of Cell Densities:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Statistical significance

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Biologicial relevance

Details on results:

Combined Limit/Range-Finding Test:

The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

At the end of the test, a concentration-related increase of both growth rate and yield inhibition was found at all test concentrations, resulting in 46% 90% inhibition of growth rate and yield, respectively, in the highest test concentration.

Based on these results, samples taken from solutions containing 10 and 100% of the SS prepared at 100 mg/L were analysed. The concentrations measured at the start of the test were 7.8 and 80 mg/L, respectively, which decreased to 0.038-0.41% of the initial concentrations at the end of the test (see also Table 2 of the appended Analytical Report).

After 48 and 72 hours of exposure, the test solution prepared at the highest concentration (i.e. the undiluted SS) was observed to be slightly hazy.

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 5.7, 11, 20, 40 and 73 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. At the end of the exposure period, the measured concentrations had decreased below the limit of detection (i.e. below 0.0049 mg/L). The concentrations measured in the samples taken from solutions with algae were comparable to the concentrations measured in the samples without algae.

Similarly to the combined limit/range-finding test, test solutions were observed to be hazy in solutions containing 32% of the SS and higher at the end of the test, whereby haziness increased with increasing test item concentration. The reason for the haziness could not be determined with certainty.

Based on the obtained results, the Time Weighted Average (TWA) concentrations were calculated and used to determine the effect parameters (see Table 4).

Mean Cell Densities:
Figure 1 shows growth curves at different TWA concentrations of Butyl Butyryl Lactate. The individual and group mean cell densities measured at 24h intervals are given in Table 10.

Inhibition of Growth Rate and Inhibition of Yield:
Table 5 shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereas Table 6 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 7 (see Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 2 and Appendix 3.

A dose-related inhibition of growth rate was observed at the end of the test, resulting in 32% inhibition at the end of the test. Statistically significant inhibition of growth rates was found at average concentrations of 2.0 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at concentrations of 6.6 mg/L and below, where the observed inhibition was below 10%.

A dose-related inhibition of yield was observed at the end of the test, resulting in 81% inhibition at the end of the test. Statistically significant inhibition of yield was found at all test concentrations.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 11 mg/L when compared to the control.

Determination of Effect Concentrations:
Table 8 shows the effect parameters based on TWA concentrations, see also Appendix 4.

Experimental Conditions:

Table 9 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 units).

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within ±1°C).

Results with reference substance (positive control):

See Appendix 5 (attached below).

Reported statistics and error estimates:

See "Any other information about materials and methods incl. tables".

Table 1
Mean Cell Densities (x10⁴Cells/mL) during the Combined Limit/Range-Finding Test

Time (h)	Butyl Butyryl Lactate;%SS prep. at 100 mg/L
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	4.9	n.d.	n.d.	2.7
48	31	n.d.	n.d.	5.1
72	164	149	141	18
n.d. – not determined.

Table 2
Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

Butyl Butyryl Lactate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.699	0.0201	6
1.0	1.668	0.0192	3	1.9
10	1.649	0.0117	3	3.0
100	0.921	0.1600	6	46

 

Table 3
Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

Butyl Butyryl Lactate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	162.9	9.65	6
1.0	148.0	8.47	3	9.2
10	139.9	4.90	3	14
100	16.8	11.31	6	90

Table 4
Time Weighted Average Versus Percentage of the Saturated Solution

Butyl Butyryl Lactate %SS prep. at 100 mg/L	Measured concentration (mg/L)			TWA conc. (mg/L)
	t=0h	t=24h	t=72 h
10	5.69	5.95	0.0025^2	2.0
18	11.1	10.6	0.0025^2	3.7
32	20.3	18.5	0.0025^2	6.6
32^1	16.4	15.0	0.0025^2	5.4
56	40.3	27.0	0.0025^2	11
100	73.1	59.1	0.0025^2	22

¹Without algae.;²Estimated as half of the LOD (LOD = 0.0049 mg/L).

Table 5
Growth Rate and Percentage Inhibition for the Total Test Period

Butyl Butyryl Lactate TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.724	0.0293	6
2.0	1.625	0.0172	3	5.8^#
3.7	1.600	0.0266	3	7.2^#
6.6	1.554	0.0138	3	9.9^#
11	1.478	0.0225	3	14*
22	1.178	0.0732	3	32*

* Effect was statistically significant.; ^#Effect was statistically significant but biologically not relevant (<10%).

Table 6
Growth Rate and Percentage Inhibition at Different Time Intervals

 

Butyl Butyryl Lactate TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.783		1.851		1.539
2.0	3	1.735	2.7	1.527	17	1.613	-4.8
3.7	3	1.728	3.1	1.587	14	1.485	3.5
6.6	3	1.689	5.3	1.501	19	1.474	4.2
11	3	1.673	6.2	1.580	15	1.181	23
22	3	1.047	41	0.648	65	1.840	-20

Table 7
Yield and Percentage Inhibition for the Total Test Period

Butyl Butyryl Lactate TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	176.0	15.38	6
2.0	130.1	6.79	3	26*
3.7	120.7	9.56	3	31*
6.6	105.0	4.37	3	40*
11	83.4	5.75	3	53*
22	33.8	7.12	3	81*

* Effect was statistically significant.

Table 8
Effect Parameters

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	<2.0	6.6	6.1	13	>22
	lower 95%-cl			5.3	12
	upper 95%-cl			7.0	14
Yield	Value	<2.0	<2.0	<2.0	2.0	8.0
	lower 95%-cl				1.8	7.5
	upper 95%-cl				2.3	8.5

cl – confidence limit;* based on statistical significance;^#based on biological relevance

. Table 9
pH Levels Recorded during the Final Test

Butyl Butyryl Lactate TWA conc. (mg/L)	pH
	t=0h	t=72h
Control	8.2	8.4
2.0	8.2	8.3
3.7	8.1	8.3
6.6	8.1	8.2
11	8.1	8.1
22	8.0	8.1

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Butyl Butyryl Lactate inhibited growth rate of this fresh water algae species significantly at TWA concentrations of 3.7 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded a TWA concentration of 22 mg/L being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L during the test period of 72 hours.

The 72h-EC50 for yield inhibition (EYC50) was 8.0 mg/L based on TWA concentrations (with a 95% confidence interval ranging from 7.5 to 8.5 mg/L).

The 72h-NOEC for growth rate inhibition was below 2.0 mg/L (TWA concentration) based on statistical significance.

The 72h-NOEC for growth rate inhibition was 6.6 mg/L (TWA concentration) based on biological relevance.

The 72h-NOEC for yield reduction was below a TWA concentration of 2.0 mg/L based on both statistical significance and biological relevance.

Executive summary:

The objective o fthe study was to evaluate Butyl Butyryl Lactate for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀ and EC₅₀ for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. 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.Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10⁴cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The concentrations measuredat the start of the testwere 5.7, 11, 20, 40 and 73 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. At the end of the exposure period, the measured concentrations had decreased below the limit of detection (i.e. below 0.0049 mg/L).

At the end of the test, a dose-related inhibition of growth rate and yield was observed at all test concentrations, resulting in 32% and 81% inhibition of growth rate and yield, respectively, at the highest test concentration.

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

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	<2.0	6.6	6.1	13	>22
	lower 95%-cl			5.3	12
	upper 95%-cl			7.0	14
Yield	Value	<2.0	<2.0	<2.0	2.0	8.0
	lower 95%-cl				1.8	7.5
	upper 95%-cl				2.3	8.5

cl – confidence limit, * - based on statistical significance,^#- based on biological relevance, n.a. – not applicable.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Butyl Butyryl Lactate inhibited growth rate and reduced yield of this fresh water algae species significantly at TWA concentrations of 2.0 mg/L and higher.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

22 mg/L

Additional information

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