Butyl anthranilate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-12-08 to 2017-12-14, with the definitive exposure phase from 2017-12-11 to 2017-12-13.

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Butyl anthranilate

EC Number:

231-816-7

EC Name:

Butyl anthranilate

Cas Number:

7756-96-9

Molecular formula:

C11H15NO2

IUPAC Name:

butyl 2-aminobenzoate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Anthranilsäurebutylester

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure (48 hours). The samples were analysed with a HPLC-DAD method, implemented under non-GLP and documented finally in the GLP raw data.

Sampling for the analytical monitoring At the start of the exposure (0 hours), samples were taken from the freshly prepared concentration levels and the control for analysis. At the end of the exposure (48 hours), samples for the analyses of the old media were taken from test replicates containing daphnids.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the initially measured concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
A stock solution (10.0 mg/L of the test item were weighed out) was prepared with dilution water (adjusted to pH 8.0 ± 0.2) three days prior to the start of the exposure (at day -3). An appropriate amount of the test item was weighed out and transferred into a glass flask with an appropriate amount of the pH-adjusted dilution water (pH 8.0 ± 0.2). This solution was stirred with a magnetic stirrer at approximately 1100 rpm for 72 hours at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour for separation of undissolved test item. Thereafter, the stock solution was removed by siphoning from the approximate center of the water body. The stock solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The stock solution was used as the highest concentration level and for the preparation of further concentration levels by dilution with dilution water.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2.2, prepared by dilution of the stock solution 10.0 mg/L with dilution water (see Table 2), were tested as follows: 0.427 - 0.939 - 2.07 - 4.55 - 10.0 mg/L
The test item concentrations were selected based on the results of a non-GLP preliminary range finding test.

Control Dilution water without test item incubated under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2°C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany


Composition of the Culture Medium according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 176

Test temperature:

18 - 22°C, constant within ± 1°C
19.0 °C

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Conductivity:

see any other information on materials and methods incl. tables

Details on test conditions:

Number of daphnids 20 daphnids, divided into 4 replicates, each with 5 daphnids, were
and replicates used for each concentration level and the control.

Age of the daphnids Less than 24 hours old daphnids from a healthy stock were used for
at the start of the exposure the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target) 18 - 22 °C, constant within ± 1 °C

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI (2.11 - 3.67)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

< 0.329 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: statsitical evaluation

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.329 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: empirical evaluation based on immobilization data

Effect concentrations 4

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI (5.63 - 7.26)

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: CI(1.27 - 3.09)

Effect concentrations 6

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 7.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 7

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

7.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017-12-06 to 2017-12-07. For results of the most recent of the monthly performed reference tests, see Table 6.

EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.64 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.00 - 4.00 mg/L

Reported statistics and error estimates:

Methods of evaluation
The EC100-values after 24 and 48 hours were empirically derived from the observation data.
The effect levels (EC10 / 50 / 100) given are based on the geometric mean measured concentrations of the test item.

EC-values and statistical analyses The EC10- and the EC50-values after 24 and 48 hours were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The concentration-effect relationships of the test item are shown graphically.
The EC50-value for the reference item and its confidence limits were calculated accordingly.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Ratesafter 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
7.46	60	60	60	60	60	100	100	100	100	100
3.28	0	20	0	40	15	60	80	40	60	60
1.41	0	0	0	0	0	40	60	20	0	30
0.650	0	0	0	0	0	20	40	40	60	40
0.329	0	0	0	0	0	20	0	0	20	10
Control 1	0	0	0	0	0	0	20	0	40	7.5^1)
Control 2	0	0	0	0	0	0	0	0	0

1)    = mean value of pooled control

Absolute Numbers of immobiledaphnids after 24 and 48 hours of Exposure in the Definitive Test

             (n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
7.46	3 / 5	3 / 5	3 / 5	3 / 5	12 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
3.28	0 / 5	1 / 5	0 / 5	2 / 5	3 / 20	3 / 5	4 / 5	2 / 5	3 / 5	12 / 20
1.41	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	2 / 5	3 / 5	1 / 5	0 / 5	6 / 20
0.650	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	1 / 5	2 / 5	2 / 5	3 / 5	8 / 20
0.329	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	1 / 5	0 / 5	0 / 5	1 / 5	2 / 20
Control 1	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	1 / 5	0 / 5	2 / 5	3 / 40^1)
Control 2	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5

 

1)    = value of pooled control

 

 

 

Additional Observations during the Definitive Test

 

All test item concentrations were visually clear throughout the exposure period. No Tyndall effect was observed in the stock solution.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item Anthranilsäurebutylester, the 48-hour EC50 for Daphnia magna was 2.01 mg/L (95% confidence limits: 1.27 - 3.09 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Anthranilsäurebutylester (batch number:1/12) were determined at the test facility according to OECD 202 (2004) from 2017-12-08 to 2017-12-14, with the definitive exposure phase from 2017-12-11 to 2017-12-13.

The study was conducted under static conditions over a period of 48 hours with a test item solution with a nominal concentration of 10.0 mg/L of the test item Anthranilsäurebutylesterand further four dilution levels prepared in a geometric series with a separation factor of 2.2(nominal concentrations of 0.427 to 10.0 mg/L).

All test item concentrations were visually clear throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrations of the test itemAnthranilsäurebutylesterwere analytically verified via HPLC-DAD in fresh media at the start (0 hours) and in old media at the end of the exposure (48 hours) in all concentration levels and in the control. Details of the analytical method are presented in section14.

The measured concentrations of the test item at test start were in the range of 73 to 86% of the nominal values. At 48 hours, the measured concentrations were in the range of 63 to 71% of the nominal values, corresponding to 80 to 90% of the initially measured concentrations in all concentration levels. The analytical results reflected the dilution factor of 2.2 between the concentration levels.The geometric mean measured concentrations were: 0.329 – 0.650 – 1.41 – 3.28 – 7.46 mg/L.

The measured test item concentrations remained stable within ±20% of the initially measured concentrations over the test period. This indicates that the test item concentrations were successfully maintained for the duration of the test. The effect concentrations given in the table below are based on the geometric mean measured concentrations of the test item Anthranilsäurebutylester.

The validity criteria of the test guideline were fulfilled.

 

 EC₁₀-, EC₅₀- (with 95% Confidence Limits) and EC₁₀₀-Values

(based on the geometric mean measured concentrations of the test item)

Effect levels		Test duration [hours]		Anthranilsäurebutylester Geometric mean measured test item concentrations [mg/L]
EC10 (with 95% confidence limits)		24		2.80		(Cl: 2.11 – 3.67)
		48		< 0.329^1)
			0.329^2)
EC50 (with 95% confidence limits)		24		6.38		(Cl: 5.63 – 7.26)
		48		2.01		(Cl: 1.27 – 3.09)
EC100		24		> 7.46
		48		7.46

 

1)    = statistical evaluation

= empirical evaluation based on mean immobilization data