Orthoperiodic acid

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2012 to 12 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A read-across justification report (RAAF) will be added to Section 13 as soon as possible.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from the control and each test group (replicates R1 – R4 pooled).
- Sampling method: Samples were taken at 0 and 48 hours for quantitative analysis. Samples at the No Observed Effect Concentration and above only were analysed.
- Sample storage conditions before analysis: Samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
An amount of test material (2200 mg) was dispersed in 11 litres of reconstituted water with the aid of a propeller stirrer at a rate of approximately 1500 rpm for a period of 24 hours. After stirring, any undissolved test material was removed using a 0.2 μm Gelman Acrocap filter (initial approximate 100 mL discarded to pre-condition the filter) to give the 100 % v/v saturated solution. Serial dilutions were then performed in reconstituted water to give the required saturated solution test concentrations. Aliquots of the saturated solution test concentrations were then each separately added to a final volume of 1 litre of reconstituted water to give the required test concentrations.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): 1st instar, less than 24 hours old
- Method of breeding: Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: Daphnia were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L (as CaCO3)

Test temperature:

20 - 21 °C

pH:

8.0 - 8.2

Dissolved oxygen:

8.7 - 9.0 mg O2/L

Nominal and measured concentrations:

0.010, 0.032, 0.10, 0.32, 1.0 % v/v saturated solution (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type : closed
- Fill volume: 200 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
- Preparation of Reconstituted water: 25 mL of CaCl2.2H2O (11.76 g/L), MgSO4.7H2O (4.93 g/L), NaHCO3 (2.59 g/L) and KCl (0.23 g/L) was added per litre deioinsed water.
- pH: 7.8 ± 0.2 adjusted (if necessary with NaOH or HCl
- Conductivity: < 5 µS/cm
- Culture medium different from test medium: yes. Culture medium was Elendt M7 medium
- Intervals of water quality measurement: Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark with 20 minute dawn/dusk transition
- Light intensity: 664 - 687 lux

EFFECT PARAMETERS MEASURED
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Range finding study (Test 1)
- Test concentrations: 0.10, 1.0, 10, 100 % v/v saturated solution (nominal)
- Results: Due to complete immobilisation in all concentrations used in the initial range-finding test it was necessary to conduct a second range-finding test.

- Range finding study (Test 2)
- Test concentrations :0.0010, 0.010, 0.10, 1.0 % v/v saturated solution (nominal)
- Results used to determine the conditions for the definitive study: no immobilisation was observed at the nominal test concentrations of 0.0010 and 0.10 % v/v saturated solution. However, immobilisation was observed at 0.10 and 1.0 % v/v saturated solution.
Based on this information nominal test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 % v/v saturated solution were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 0.098 - 0.33 mg/L (95 % CL)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.099 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Immobilisation data: None of the Daphnia in the control, 0.010, 0.032 and 0.10 % v/v saturated solutions were immobilised during the 48 hour exposure period. However, all of the Daphnia in the 0.32 and 1.0 % v/v saturated solutions were immobilised within 24 hours of exposure (see Table 3)
- Observations: The test preparations were observed to be clear colourless solutions for the duration of the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.
- EC50/LC50: 48 hour EC50 0.45 mg/L (95 % C.I. 0.42-0.48 mg/L)

Reported statistics and error estimates:

The EC50 value and associated confidence limits at 24 and 48 hours were calculated using the geometric mean method using the ToxCalc software package.

Table 1: Cumulative Immobilisation Data in the Initial Range-finding Test

Nominal conc. (% v/v saturated solution)	Cumulative immobilised Daphnia (initial population: 10 per replicate)
	24 hours	48 hours
Control	1	*
0.10	10	*
1.0	10	*
10	10	*
100	10	*

*The test was stopped at 24 hours after exposure due to 100 % immobilisation in all test concentrations.

Table 2: Cumulative Immobilisation Data in the Second Range-finding Test

Nominal conc. (% v/v saturated solution)	Cumulative immobilised Daphnia (initial population: 10 per replicate)
	24 hours	48 hours
Control	0	0
0.0010	0	0
0.010	0	0
0.10	3	5
1.0	10	10

Table 3: Cumulative Immobilisation Data in the Definitive Test

Nominal conc. (% v/v saturated solution)	Cumulative immobilised Daphnia (initial population: 5 per replicate)
	24 hours					48 hours
	R1	R2	R3	R4	Total	R1	R2	R3	R4	Total
Control	0	0	0	0	0	0	0	0	0	0
0.010	0	0	0	0	0	0	0	0	0	0
0.032	0	0	0	0	0	0	0	0	0	0
0.10	0	0	0	0	0	0	0	0	0	0
0.32	5	5	5	5	20	5	5	5	5	20
1.0	5	5	5	5	20	5	5	5	5	20

R1 - R4 = Replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

Based on the mean-measured test concentrations of the test media, the 48-hour EC50 was 0.18 mg/L with 95 % confidence limits of 0.098 – 0.33 mg/L and the No Observed Effect Concentration was 0.098 mg/L.

Executive summary:

The acute toxicity of the test material to Daphnia magna was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 202 and EU Method C.2 under static conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 % v/v saturated solution for 48 hours. The number of immobilised Daphnia were recorded after 24 and 48 hours.

The No Observed Effect Concentration and above only were analysed. Analysis of the freshly prepared media at 0 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.098, 0.34 and 0.91 mg/L, respectively. Analysis of the old media at 48 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.099, 0.32 and 1.1 mg/L. It was considered appropriate to base the results on mean-measured test concentrations.

The 48-hour EC50 for the test item to Daphnia magna, based on mean-measured test concentrations, was 0.18 mg/L with 95 % confidence limits of 0.099 – 1.0 mg/L. The Lowest Observed Effect Concentration was considered to be 0.33 mg/L. The No Observed Effect Concentration was 0.099 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

A read-across justification report (RAAF) will be added to Section 13 as soon as possible.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 0.098 - 0.33 mg/L (95 % CL)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.099 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 October 2018 to 21 December 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:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: At t=0 h and t=48 h
Volume: 4.8 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 4.8 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:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of test material tested was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test material.
Preparation of test solutions started with the highest concentration of 100 mg/L in the combined limit/range-finding test and 1.0 mg/L in the final test. No other treatment than vigorous shaking was needed to completely dissolve the test material in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colourless at the end of the preparation procedure.
Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Justification for species other than prescribed by test guideline: This system has been selected as an internationally accepted invertebrate species.
- Source: In-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions. 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.
- Age of parental stock: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.
- Feeding during test: No feeding


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 to reach the following concentrations:
Salts: 0.71 mg/L H3BO3, 0.25 mg/L FeSO4.7H2O, 0.090 mg/L MnCl2.4H2O, 0.076 mg/L LiCl, 0.018 mg/L RbCl, 0.038 mg/L SrCl2.6H2O, 0.015 mg/L Na2MoO4.2H2O, 0.0040 mg/L NaBr, 0.0042 mg/L CuCl2.2H2O, 0.013 mg/L ZnCl2, 0.010 mg/L CoCl2.6H2O, 0.0032 mg/L KI, 0.0022 mg/L Na2SeO3, 0.00057 mg/L NH4VO3, 0.62 mg/L Na2EDTA.2H2O, 7.5 mg/L Na2SiO3.5H2O, 0.27 mg/L NaNO3, 0.14 mg/L KH2PO4, 0.18 mg/L K2HPO4
Vitamins: 75.0 µg/L Thiamine hydrochloride, 1.0 µg/L B12, 0.75 µg/L Biotin.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19 - 20 °C

pH:

7.9 - 8.2

Dissolved oxygen:

9.6 - 9.9 mg/L

Nominal and measured concentrations:

0.022, 0.046, 0.10, 0.22 and 0.46 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Material, size, fill volume: Glass, 50 mL
- Aeration: No aeration of the test solutions was applied.
- No. of organisms per vessel: 20 per concentration
- No. of vessels per concentration: 4
- No. of vessels per vehicle control: 4
- Biomass loading rate: 5 per vessel containing 50 mL of test solution.
- Introduction of daphnids: 12 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): 211.5 mg/L CaCl2.2H2O, 88.8 mg/L MgSO4.7H2O, 46.7 mg/L NaHCO3, 4.2 mg/L KCl.
- Hardness: The hardness of test medium expressed as CaCO3: 180 mg/L
- pH of test medium: pH between 6 and 9.

OTHER TEST CONDITIONS
- Photoperiod: A daily photoperiod of 16 hours.

EFFECT PARAMETERS MEASURED:
- Immobility: At 24 hours and at 48 hours.
- pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.
- Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

VEHICLE CONTROL PERFORMED: Test medium without test material or other additives.

RANGE-FINDING STUDY
- Test concentrations: 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 a concentration of 100 mg/L. 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 0.10, 1.0 and 10 mg/L in the combined range-finding test.
- Results used to determine the conditions for the definitive study: At the end of the test, 70 % immobility was observed at the lowest test concentration while 100 % immobility was observed at higher concentrations. Therefore, the expected EC50 was below nominal 0.10 mg/L.
Based on these results, samples taken from nominally 0.10 and 1.0 mg/L and the control were analysed. The measured concentrations at the start of the test were 0.091 and 1.2 mg/L, respectively. During the exposure period, the concentrations changed to 115 and 74 % of initial at the end of the test.
A small response was observed in the control, which was probably caused by carry over. It will have no impact on the results and conclusions of the study.
All test conditions were maintained within the limits prescribed by the study plan.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.086 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence interval between 0.066 and 0.11 mg/L

Details on results:

- Measured Concentrations: Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.017, 0.035, 0.070, 0.16 and 0.34 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 93 - 101 % relative to initial at the end of the test.
It should be noted that small responses were detected in the control throughout the test. These responses were probably caused by carry over. Considering that the concentration could be estimated only by extrapolation of the calibration curve and was 5.3 – 7.0 % of the concentration measured at the lowest test concentration, this was considered negligible.
Based on these results, effect parameters were expressed as initially measured concentrations.
- Immobility: No immobility was observed in the control. At the end of the test, a dose-related increase of immobility was observed at 0.035 mg/L and higher, reaching 100 % immobility at the highest test concentration.
The responses recorded in this test allowed for reliable determination of an EC50 and were in agreement with what was expected based on the results of the combined limit/range-finding test.

Results with reference substance (positive control):

The objective of the study was to evaluate potassium dichromate (K2Cr2O7) 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 48 hours of exposure (Charles River Den Bosch Study Number 20174202).
The reference test was carried out to check the sensitivity of the test system as used by Charles River Den Bosch. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
The reference material, potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K50664264) was obtained from Merck, Darmstadt, Germany.
The study met the acceptability criteria prescribed by the study plan and was considered valid.
The actual responses in this reference test with K2Cr2O7 are generally within the ranges of the expected responses at the different concentrations, i.e. the 48 h EC50 was between 0.28 and 0.90 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.
The 48 h EC50 was 0.40 mg/L with a 95 % confidence interval between 0.33 and 0.48 mg/L.

Reported statistics and error estimates:

The 24 and 48 h-EC50-values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test material concentrations (initially measured) using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

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

Time (h)	Replicate	Test Material Measured Conc.  (mg/L)
		Control	0.017	0.035	0.070	0.16	0.34
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	2	3	5
	B	0	1	0	5	5	5
	C	0	0	4	2	4	5
	D	0	0	4	2	5	5
	Total immobilised	0	1	8	11	17	20
	Effect %	0	5	40	55	85	100
48	A	0	0	0	1	3	5
	B	0	0	0	5	5	5
	C	0	0	4	1	4	5
	D	0	0	4	2	5	5
	Total immobilised	0	0	8	9	17	20
	Effect %	0	0	40	45	85	100

 

Parameters of the Probit Analysis at 24 h

Parameter	Value
Computation runs:	6.00000
Slope b:	2.55404
Intercept a:	2.85870
Variance of b:	0.17547
Goodness of Fit
Chi²:	2.57327
Degrees of freedom:	3.00000
p(Chi²):	0.46200
Log LC50:	-1.11929
SE Log LC50:	0.06205
g-Criterion:	0.10333
F:	43.34000
p(F) (df: 1;3):	0.00700

 

Results of the Probit Analysis at 24 h

Parameter	EC50
Value [mg/L]	0.076
lower 95 %-cl	0.057
upper 95 %-cl	0.101

 

Parameters of the Probit Analysis at 48 h

Parameter	Value
Computation runs:	6.00000
Slope b:	2.74304
Intercept a:	2.92758
Variance of b:	0.19428
Goodness of Fit
Chi²:	6.17135
Degrees of freedom:	3.00000
p(Chi²):	0.10400
Log LC50:	-1.06727
SE Log LC50:	0.05905
g-Criterion:	0.09919
F:	18.82700
p(F) (df: 1;3):	0.02300

 

Results of the Probit Analysis at 48 h

Parameter	EC50
Value [mg/L]	0.086
lower 95 %-cl	0.066
upper 95 %-cl	0.112

Acute Immobilisation of Daphnia After 24 and 48 Hours in the Reference Test with Potassium Dichromate

Concentration K2Cr2O7 (mg/L)	Number Exposed	% immobile		Expected response (%) After 48 hours *
		24h	48h	Minimal	Maximal
Control	20	0	0	0	10**
0.10	20	0	0	0	10
0.18	20	0	15	0	10
0.32	20	0	15	0	30
0.56	20	15	75	0	100
1.0	20	95	100	40	100
1.8	20	100	100	100	100

* Based on historical data of the previous years (n > 60).

** A maximum response of 10 % does not invalidate the results of the test.

Analytical Results

Time of Sampling (h)	Date of Sampling	Date of Analysis*	Concentration (mg/L)		Relative to Nominal [%]	Relative to Initial [%]
			Nominal	Analysed
0	10 December 2018	20 December 2018	0	0.0012**	n.a
			0.022	0.0172	78
			0.046	0.0354	77
			0.10	0.0703	70
			0.22	0.160	73
			0.46	0.342	74
48	12 December 2018	20 December 2018	0	0.00092**	n.a	n.a
			0.022	0.0174	79	101
			0.046	0.0354	77	100
			0.10	0.0674	67	96
			0.22	0.148	67	93
			0.46	0.331	72	97

*Samples were stored in the freezer (≤ -15 °C) until the sample preparation on 19 Dec 2018. Due to acquisition problems the samples were analysed on 20 Dec 2018. The samples were stored at room temperature.

** Estimated value, calculated by extrapolation of the calibration curve. This is probably caused by carry over since a similar response was observed in the analytical blank.

n.a. Not applicable.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48 h EC50 for Daphnia magna exposed to the test material was 0.086 mg/L based on measured concentrations (95 % confidence interval between 0.066 and 0.11 mg/L).

Executive summary:

The acute toxicity to daphnia was assessed according to OECD guideline No. 202 and in compliance with GLP. The objective of the study was to evaluate the test material 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 batch of the test material tested was a white crystalline powder with a purity of 99.49 % and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 0.022, 0.046, 0.10, 0.22 and 0.46 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 measured concentrations at the start of the test were 0.017, 0.035, 0.070, 0.16 and 0.34 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 93 - 101 % relative to initial at the end of the test

No immobility was observed in the control. At the end of the test, a dose-related increase of immobility was observed at 0.035 mg/L and higher, reaching 100 % immobility at the highest test concentration.

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

In conclusion, the 48 h EC50 for Daphnia magna exposed to the test material was 0.086 mg/L based on measured concentrations (9 5% confidence interval between 0.066 and 0.11 mg/L) and was already reached after 24 hours of exposure.

Description of key information

Key Study:

Under the conditions of the study the 48-h EC50 for Daphnia magna exposed to the test material was 0.086 mg/L based on measured concentrations (95 % confidence interval between 0.066 and 0.11 mg/L).

Read-Across Substance:

The 48-hour EC50 for the test material to Daphnia magna based on mean-measured test concentrations was 0.18 mg/L with 95 % confidence limits of 0.099 – 1.0 mg/L.  

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.086 mg/L

Additional information

Key Study (Vromans, 2019)

The acute toxicity to daphnia was assessed according to OECD guideline No. 202 and in compliance with GLP. 

The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as set forth by Klimisch et al. (1997).

The objective of the study was to evaluate the test material 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 batch of the test material tested was a white crystalline powder with a purity of 99.49 % and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 0.022, 0.046, 0.10, 0.22 and 0.46 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 measured concentrations at the start of the test were 0.017, 0.035, 0.070, 0.16 and 0.34 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 93 - 101 % relative to initial at the end of the test

No immobility was observed in the control. At the end of the test, a dose-related increase of immobility was observed at 0.035 mg/L and higher, reaching 100 % immobility at the highest test concentration.

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

In conclusion, the 48 h EC50 for Daphnia magna exposed to the test material was 0.086 mg/L based on measured concentrations (9 5% confidence interval between 0.066 and 0.11 mg/L) and was already reached after 24 hours of exposure.

Read-Across Substance (Parr, 2013)

The acute toxicity of the test material to Daphnia magna was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 202 and EU Method C.2 under static conditions. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as set forth by Klimisch et al. (1997).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 % v/v saturated solution for 48 hours. The number of immobilised Daphnia were recorded after 24 and 48 hours.

The No Observed Effect Concentration and above only were analysed. Analysis of the freshly prepared media at 0 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.098, 0.34 and 0.91 mg/L, respectively. Analysis of the old media at 48 hours for the 0.10, 0.32 and 1.0 % v/v saturated solution test concentrations showed measured concentrations of 0.099, 0.32 and 1.1 mg/L. It was considered appropriate to base the results on mean-measured test concentrations.

The 48-hour EC50 for the test item to Daphnia magna, based on mean-measured test concentrations, was 0.18 mg/L with 95 % confidence limits of 0.099 – 1.0 mg/L. The Lowest Observed Effect Concentration was considered to be 0.33 mg/L. The No Observed Effect Concentration was 0.099 mg/L.