IPEMA

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:

2021-09-21 to 2021-10-28

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:

Adopted: 2004-04-13

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test item concentrations and control (at test start and after 24 hours from fresh and aged solutions and after 48 hours from aged solutions)
- Sample storage conditions before analysis: refridgerated (1 - 10 °C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 60 minutes stirring of stock solution at 100 mg/L and 750 rpm
- Controls: Test medium control
- Concentration of final test solutions: 100, 45.5, 20.7, 9.39 and 4.27 mg/L and control
- Test concentration separation factor: 2.2
- Evidence of undissolved material: Before stirring, small oily droplets were visible. Afterwards the solution was clear and transparent, but slightly foaming.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Strauß
- Strain, Daphnia magna (Crustacea, Cladocera) (Strauß, 1820), clone: V
- Age at study initiation: young daphnia with age < 24 h
- Method of breeding: Daphnia magna was bred as single culture (1 daphnid per 100 mL) in test medium (a mix of dechlorinated drinking water and deionised water). The pH-value was within a range of 6.0 – 9.0. The dissolved oxygen was above 60 % saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The animals were fed with single cell green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus) at least three times a week.
- Source: Federal Environment Agency in Berlin/Germany
- Feeding during test: None

Test type:

semi-static

Water media type:

freshwater

Remarks:

Elendt M4 test medium

Limit test:

no

Total exposure duration:

48 h

Hardness:

13°dH (232 mg/L CaCO3)

Test temperature:

19.8 – 20.3 °C

pH:

7.61 – 8.00

Dissolved oxygen:

= 7.28 mg/L

Nominal and measured concentrations:

Nominal: 100, 45.5, 20.7, 9.39, 4.27 mg/L
Measured (geometric mean): 76.1, 29.9, 13.6, 4.44, 1.86 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Schott flasks with lid
- Type: closed
- Fill volume: 100 mL Schott flasks with lid per treatment, each filled with as small as possible headspace
- Volume of solution: ~ 130 mL
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4, one additional replicate for physico - chemical measurements without test organisms
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 test medium
- Culture medium different from test medium: yes (a mix of dechlorinated drinking water and deionised water used as culture medium)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod /8 hours darkness daily
- Light intensity: 1300 lux (mean)

VEHICLE CONTROL PERFORMED: no

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

9.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

11.1 mg/L

95% CI:

8.43 - 14

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

19.2 mg/L

95% CI:

15.7 - 23.2

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Mortality of control: None
- Immobilisation of control: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All solutions appeared to be clear and colourless, but initial measured content of IPEMA was between 50 % and 78 % of nominal and is comparable to the measured content from non-GLP pre-tests.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ECx: between 0.6 and 2.1 mg/L (only these 2 concentrations tested)

Reported statistics and error estimates:

The 24 h and 48 h EC50 are the calculated concentrations where 50 % of the daphnids were
immobilised after 24 and 48 hours, respectively.
Based on nominal concentrations, the value for EC50 after 24 hours was determined by probit
analysis using linear maximum likelihood regression and the EC50 after 48 hours was determined
by Weibull analysis using linear maximum likelihood regression.
Based on mean measured concentrations, the value for EC50 after 24 and 48 hours was
determined by probit analysis using linear maximum likelihood regression.
The evaluation of data was performed by ToxRat Professional 3.3.0. ToxRat Professional, Version
3.3.0 performs calculations with unrounded values.
The NOEC was established based on the highest concentration at which the immobilisation is not
higher than the allowed control immobilisation (= 10 % immobilisation).

Validity Criteria of the Study

Control immobilisation: The percentage of immobilisation should be ≤ 10 %. In this study the control immobilisation was 0 %.

Oxygen concentration: The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in all test units. In this test, the dissolved oxygen concentration at the end of the test was ≥ 7.28 mg/L.

Table 1:       Results of the test, 24 h values

 

	Nominal test item concentration [mg/L]
	Control	4.27	9.39	20.7	45.5	100
	Immobilised daphnids after 24 h
Replicate 1	0	0	0	2	4	5
Replicate 2	0	0	0	4	4	5
Replicate 3	0	0	0	2	4	5
Replicate 4	0	0	0	2	5	5
∑	0	0	0	10	17	20
%	0	0	0	50	85	100

Table 2:       Results of the test, 48 h values

 

	Nominal test item concentration [mg/L]
	Control	4.27	9.39	20.7	45.5	100
	Immobilised daphnids after 48 h
Replicate 1	0	0	0	3	5	5
Replicate 2	0	0	0	4	5	5
Replicate 3	0	0	0	2	5	5
Replicate 4	0	0	1	3	5	5
∑	0	0	1	12	20	20
%	0	0	5	60	100	100

 

Table 3:       Temperature of the test solutions

	Nominal test item concentration [mg/L]
	Control	4.27	9.39	20.7	45.5	100
Time [h]	Temperature [°C]
0 fresh	20.3	20.1	20.1	20.2	20.2	20.2
24 aged	20.2	20.2	20.2	20.3	20.3	20.3
24 fresh	19.9	20.0	19.9	19.8	19.8	19.9
48 aged	20.1	20.1	20.1	20.1	20.1	20.1
Mean	20.1	20.1	20.1	20.1	20.1	20.1
Std. dev.	0.2	0.1	0.1	0.2	0.2	0.2
Mean	20.1
Std. dev.	0.2

 

Table 4:       pH-values of the test solutions

	Nominal test item concentration [mg/L]
	Control	4.27	9.39	20.7	45.5	100
Time [h]	pH
0 fresh	7.92	8.00	8.00	8.00	8.00	7.99
24 aged	7.86	7.61	7.68	7.74	7.74	7.79
24 fresh	7.84	7.99	8.00	8.00	7.98	7.99
48 aged	7.87	7.85	7.81	7.80	7.80	7.83
Mean	7.87	7.86	7.87	7.89	7.88	7.90
Std. dev.	0.03	0.18	0.16	0.14	0.13	0.11
Mean	7.88
Std. dev.	0.12

 

Table 5:       O2 concentration of the test solutions

	Nominal test item concentration [mg/L]
	Control	4.27	9.39	20.7	45.5	100
Time [h]	Oxygen [mg/L]
0 fresh	8.73	8.71	8.69	8.68	8.71	8.69
24 aged	8.18	7.28	7.99	8.55	8.45	8.56
24 fresh	8.78	8.63	8.59	8.60	8.62	8.61
48 aged	8.73	8.18	8.21	8.55	8.60	8.60
Mean	8.61	8.20	8.37	8.60	8.60	8.62
Std. dev.	0.28	0.66	0.33	0.06	0.11	0.05
Mean	8.50
Std. dev.	0.33

 

Table 6:       Composition of Elendt M4 test medium

Stock solution	Concentration of stock solution		Amount (mL) of stock solution for 60 L final medium	Concentrations in final medium mg/L
Calcium chloride	CaCl2 × 2 H2O	588.16 g/L	30	294
Magnesium sulfate	MgSO4 × 7 H2O	246.6 g/L	30	123
Sodium hydrogencarbonate	NaHCO3	81.0 g/L	48	64.8
Potassium chloride	KCl	11.6 g/L	30	5.80
	MnCl2 × 4 H2O	21.63 g/L		0.3605
	LiCl	18.36 g/L		0.306
	RbCl	4.26 g/L		0.071
Cation solution	SrCl2 × 6 H2O	9.12 g/L	1	0.152
	CuCl2 × 2 H2O	710.8 mg/L		0.0118
	ZnCl2	780 mg/L		0.0130
	CoCl2 × 6 H2O	600 mg/L		0.0100
	NaNO3	3.29 g/L		0.274
	H3BO3	34.31 g/L		2.86
	NaBr	0.192 g/L		0.0160
Anion solution	Na2MoO4 × 2 H2O	0.738 g/L	5	0.0615
	KI	39 mg/L		0.00325
	Na2SeO3	26.3 mg/L		0.00219
	NH4VO3	6.9 mg/L		0.000575
Silica solution	Na2 SiO3 × 9 H2O	120 g/L	5	10.0
EDTA- Ferric sulphate solution	Titriplex III × 2 H2O FeSO4 × 7 H2O	5 g/L 1.991 g/L	30	2.50
				0.996
Phosphate solution	KH2PO4 K2HPO4	8.58 g/L 11.14 g/L	1	0.143
				0.184
Vitamine solution	Thiamindihydrochloride Cyanocobalamine (B12) Biotine	4.5 g/L 60 mg/L 45 mg/L	1	0.0750
				0.00100
				0.000750

 

Table 7:       Results of the toxic reference test, started on 19 October 2021

K2Cr2O7 [mg/L]	24 h		48 h
	0.6	2.1	0.6	2.1
	immobilised daphnids
Replicate 1	0	5	0	5
Replicate 2	0	5	0	5
Replicate 3	0	5	0	5
Replicate 4	0	5	0	5
∑	0	20	0	20
%	0	100	0	100

 

Table 8:       Determined concentration of IPEMA

Test item nominal [mg/L]	IPEMA nominal [mg/L]	Sampling	IPEMA found		Mean measured test item conc. [mg/L]
			[mg/L]	% of nominal
Control	0	0 h fresh	< LOD	-	-
Control	0	24 h fresh	< LOD	-	-
Control	0	48 h aged	< LOD	-	-
4.27	4.25	0 h fresh	3.19	75	1.86
4.27	4.25	24 h fresh	2.21	52	1.86
4.27	4.25	48 h aged	1.27	30	1.86
9.39	9.34	0 h fresh	6.12	66	4.44
9.39	9.34	24 h fresh	5.74	61	4.44
9.39	9.34	48 h aged	3.3	35	4.44
20.7	20.6	0 h fresh	16.1	78	13.6
20.7	20.6	24 h fresh	15.2	74	13.6
20.7	20.6	48 h aged	11.6	56	13.6
45.5	45.3	0 h fresh	22.7	50	29.9
45.5	45.3	24 h fresh	25.8	57	29.9
45.5	45.3	48 h aged	36.2	80	29.9
100	99.5	0 h fresh	59.4	60	76.1
100	99.5	24 h fresh	60	60	76.1
100	99.5	48 h aged	96	96	76.1

- = not calculated; LOD = 0.128 mg/L IPEMA, LOQ = 0.427 mg/L IPEMA

Validity criteria fulfilled:

yes

Conclusions:

According to the results of the test, the EC50 (48 h) for immobilisation was determined to be
19.2 mg/L (nominal) and 11.1 mg/L (mean measured). The NOEC (48 h) was set at 9.39 mg/L (nominal) and 4.44 mg/L (mean measured).

Description of key information

According to the results of the test, the EC50 (48 h) for immobilisation was determined to be 19.2 mg/L (nominal) and 11.1 mg/L (mean measured). The NOEC (48 h) was set at 9.39 mg/L (nominal) and 4.44 mg/L (mean measured).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

mean measured

Effect concentration:

11.1 mg/L