AERO 5100 PROMOTER

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:

25 - 27 Aug 1998

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)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and solvent control.
- Sampling method: At 0 h, additional duplicate vessels (280 mL) were prepared in an identical manner to those used in the test and their contents analysed. After 48 h, the contents of the vessels used in the test were analysed

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An aliquot (50 mg) of test substance was dissolved in 50 mL of ethanol to give an initial stock solution of 1 mg/mL. Serial dilutions of this stock solution were prepared with ethanol. Due to the volatility of the test substance (100% volatile by weight) an "air-free" test design was employed. Bottles of a total volume of 280 mL with air-tight screw caps, were filled to capacity with Elendt M4 medium and the caps secured. Each bottle was inverted and using a syringe to puncture a Teflon coated disc, located in the centre of the bottle cap, was spiked with 28 µL of the appropriate stock solution (to give a solvent loading equivalent to 100 µL/L). Each bottle was then shaken vigorously to aid dispersion.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): ethanol
- Controls: yes, test medium and solvent control
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house culture, obtained from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France
- Age at study initiation: first instar; less than 24 h old (The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals were used in the test)
- Method of breeding: Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 800 mL of Elendt M4 culture medium with a 16 h light: 8 h dark photoperiod at 20 ± 2 °C. Cultures were fed daily with a suspension of the unicellular green alga, Chlorella vulgaris. Culture conditions ensure that cultures reproduce by parthenogenesis.
- Feeding during test: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19 °C (at test start and end)

pH:

7.5 - 8.3 (at test start)
6.7 - 7.3 (at test end)

Dissolved oxygen:

7.4 - 9.0 mg O2/L (at test start)
6.1 - 8.0 mg O2/L (at test end)

Nominal and measured concentrations:

control, solvent control, 0.10, 0.22, 0.46, 1.0, 2.2, 4.6, 10 and 22 µg/L (nominal)
not detected, < LOQ,

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed (by aid of air-tight screw caps)
- Material, size, headspace, fill volume: 280 mL glass bottles filled to capacity with no headspace
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4 (containing 100 µL solvent/L)
- Biomass loading rate: 56 mL test solution per organism

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
Trace elements (mg/L):
H3BO3: 2.86
MnCl2.4H2O: 0.36
LiCl: 0.31
RbCl: 0.071
SrCl2.6H2O: 0.152
NaBr: 0.016
Na2MoO4.2H2O: 0.063
CuCl2.2H2O: 0.017
ZnCl3: 0.013
CoCl2.6H2O: 0.010
KI: 0.0033
Na2.SeO3: 0.0022
NH4VO3: 0.00058
Fe-EDTA solution: 3.50

Macro nutrients (mg/L):
CaCl2.2H2O: 294
MgSO4.7H2O: 123
KCl: 5.80
NaHCO3: 64.8
Na2.SiO3.9H2O: 10.0
NaNO3: 0.274
KH2PO4: 0.143
K2HPO4: 0.184

Vitamins (mg/L):
Thiamine hydrochloride: 0.075
Cyanocobalamine (Vitamin B12): 0.0010
Biotin (Vitamin H): 0.00075

- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, pH, and dissolved oxygen levels were recorded in each vessel at the start and the end of the study

OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8 h dark

EFFECT PARAMETERS MEASURED: Immobilisation, observed at 24 and 48 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2.2
- Range finding study: yes
- Test concentrations: 0.01 to 100 µg/L
- Results used to determine the conditions for the definitive study: The NOEC was approximately 0.1 µg/L and the LOEC resulting in 100% immobilisation was approximately 100 µg/L.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.007 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 0.0032 - 0.015 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Immobilisation of control: 5% (test medium control), 0% (solvent control)

Reported statistics and error estimates:

EC50 values and 95% confidence limits were calculated using the Thompson and Weil model (Thompson, W.R. and Weil, C.S. (1952) Biometrics 8: 51 -54).

Analytical measurements:

Analysis of the 4.6, 10 and 22 µg/L test concentrations gave measured concentrations ranging from 144 - 107% of nominal at 0 hours and 91 - 83% of nominal at 48 h. Due to the limited sensitivity of the method of analysis (limit of quantification >4 µg/L) it was not possible to verify the aqueous test concentrations below 4.6 µg/L. However, the stock solutions used to prepare all the exposure concentrations were analysed and found to be 97 - 72% of nominal at 0 h, so it is reasonable to infer that near nominal concentrations were achieved at the start of the study.

 

Table 1: Cumulative immobilisation of Daphnia exposed for 48 hours

Nominal concentration [µg/L]	Geometric mean measured concentration [µg/L]	Cumulative immobilised Daphnia magna (initial population: 5 per replicate)
		24 h						48 h
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	-	0	0	0	0	0	0	0	0	0	l	1	5
Solvent Control	ND	0	0	0	0	0	0	0	0	0	0	0	0
0.10	*	0	0	0	0	0	0	1	1	0	0	2	10
0.22	*	0	0	0	0	0	0	0	0	0	1	1	5
0.46	*	0	0	0	1	1	5	0	1	0	2	3	15
1.0	*	0	0	0	2	2	10	1	0	0	3	4	20
2.2	*	0	0	1	0	1	5	1	0	2	2	5	25
4.6	5.1	1	1	1	0	3	15	J	3	2	2	10	50
10	9.5	1	1	3	2	7	35	5	2	3	2	12	60
22	22	2	1	2	3	8	40	4	2	5	5	16	80

R1 -R4: Replicate No.

*: Below limit of quantification of 4 µg/L

ND: None detected

Tables 2 to 5: Summary of analytical chemistry results

Table 2: Analytical results

Occasion	Nominal concentration [µg/L]	Measured concentration [µg/L]	Expressed as a percentage of nominal
0 h	Control	ND	-
	4.6	6.634	144
	10	10.94	109
	22	23.59	107
48 h	Control	ND	-
	4.6	3.924	85.3
	10	8.328	83.3
	22	20.04	91.1

Table 3: Stock solutions

Nominal concentration [mg/mL]	Measured concentration [mg/mL]	Expressed as a percentage of nominal
1.0	0.7207	72.1
2.2	1.669	75.9
4.6	4.193	91.2
10	9.246	92.5
22	20.66	93.9
46	43.69	95.0
100	97.43	97.4
220	200.1	91.0
1000	954.8	95.5

Table 4: Procedural recoveries (test levels)

Occasion	Fortified concentration (µg/L)	Recovery as a percentage of fortified
0 h	Control	ND
	10.88	92.9
	10.88	93.9
48 h	Control	ND
	10.88	91.9
	10.88	94.8
Mean (RSD)		93.3 (1.3)

Table 5: Procedural recoveries (stock levels)

Fortified concentration (mg/mL)	Recovery as a percentage of fortified
21.76	101
21.76	97.7
Mean	99.1

Description of key information

EC50 (48 h): 0.0068 mg/L (nominal, OECD 202)

Additional information

One experimental study is available investigating the short-term effects of the substance to aquatic invertebrates (Cytec Industries, 1998). The study was performed according to OECD 202 (GLP) using Daphnia magna as test organism. A stock solution of 100 mg/L was prepared in ethanol to prepare the final nominal test solutions of 0.10, 0.22, 0.46, 1.0, 2.2, 4.6, 10 and 22 µg/L. In addition a test medium control and solvent control were tested. The actual test concentrations were measured by GC/FID analysis. Since the Limit of Quantification was < 4 µg/L only the three highest concentrations could be analysed. The analysis resulted in a mean concentration of 5.1, 9.5, and 22 µg/L (compared to nominal 4.6, 10 and 22 µg/L). Thus, the substance is considered to be stable under testing conditions. Immobilisation of Daphnids was observed resulting in an EC50 (48 h) of 0.0068 mg/L (95% CI: 0.0032 - 0.015 mg/L) based on nominal concentrations.