Pyridinium dichromate

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:

from 2010-10-05 to 2010-11-18

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Following nominal concentrations were measured: 0.00, 0.13, 0.25, 0.50, 1.00, 2.00, 4.00 and 8.00 mg/L
- Sampling method: Actual concentrations of the test substance in the test media were determined in the surplus of the test media at the beginning of the test and in the pooled test media of the replicates at the end of the test.
- Sample storage conditions before analysis: deep frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A first stock solution (ST1) with a nominal test substance concentration of 800 mg/L was prepared at the start of the test by dissolving 82.2 mg test substance in 100 mL reconstituted water according to ISO 6341 by manual homogenisation. To obtain the final stock solution which was used for the test (ST2, nominal 8 mg/L), 3 mL of ST1 were filled up to 300 mL reconstituted water. The test media were prepared by diluting the ST2 with reconstituted water.
- Controls: Blank control performed with reconstituted water only (ISO6341) without test substance
- Evidence of undissolved material: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Neonates of Daphnia magna (Crustacea, Cladocera), were obtained by hatching ephippia, supplied as "Daphtoxkit F TM magna" (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM).
- Age at study initiation : less than 24 hours old.
- Method of breeding: Neonates were obtained by hatching ephippia. The ephippia were hatched in the laboratory in a petri dish containing reconstituted water in a temperature controlled room at approx. 20 - 22 °C under continuous illumination of approximately 6 000 Lux. According to MicroBioTests Inc. the largest hatching occurs between 72 and 80 hours and the Daphnia must be collected at the latest 90 hours after initiation of hatching. The neonates were collected for this test at approximately 82 hours after initiation of hatching.
- Feeding during test: none.

ACCLIMATION
- Acclimation period: 82 hours
- Acclimation conditions; Same as the test
- Feeding during acclimation perios: Approximately 1 hour before the start of the test the neonates were fed with a suspension of Spirulina powder (MicroBioTests Inc.), as also in traditional Daphnia cultures the neonates have the opportunity to take up some feed particles.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

250 mg CaCO3/L

Test temperature:

18 - 19 °C

pH:

The pH of the relevant test media ranged from 7.6 to 7.9.

Dissolved oxygen:

At least 8.8 mg/L

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentrations: 0.00, 0.13, 0.25, 0.50, 1.00, 2.00, 4.00, 8.00 mg/L
Measured concentrations at start of test: Measured concentrations at end of test: Geometric mean concentrations (0-48 h):

Details on test conditions:

TEST SYSTEM
- Test vessel: Disposable multiwell test plates with 30 test wells with a transparent lid, provided with the Daphtoxkit F. Each plate has four 15 mL wells for each of the control and the concentrations tested. Additionally, for each concentration the plates are provided with "rinsing wells" to prevent dilution of the test substance during the transfer of the daphnia from the hatching petri dish to the test wells.
- Material, size, headspace, fill volume: 15 mL polycarbonate wells (according to Microbiotest), filled with 10 mL medium.
- Aeration: No extra aeration, but slight circulation of air is still possible when the test plate is closed.
- 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: The stock solutions for preparation of the reconstituted water according to ISO 6341 were obtained by MicroBioTests Inc. The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using.
- Culture medium different from test medium: no.
- Intervals of water quality measurement: Temperature was measured daily. Oxygen and pH at the start and the end of the test. Total hardness before the start.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 4 a.m. to 8 p.m.
- Light intensity: not applicable.

EFFECT PARAMETERS MEASURED
24 and 48 hours after the start of the exposure to the test substance the neonates were observed for their swimming behaviour. Animals not able to swim within approximately 15 s after gentle agitation of the test container were evaluated as immobilised. Animals trapped at the surface were counted, to evaluate a possible influence of the test substance to the water surface tension. Only the mobile daphnia trapped on the water surface were regarded as trapped but not the immobilised ones.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: Test concentrations: A static non GLP range finding study was conducted using 20 Daphnia per concentration and nominal concentrations of 100, 10, 1, and 0.1 mg/L.
- Results used to determine the conditions for the definitive study: This test revealed 100, 100, 40 and 0 % immobilisation respectively after 48 hours. Therefore nominal concentrations between 0.13 and 8.0 mg/L were chosen for the main experiment.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.91 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL: lower: 0.72 mg/L, upper: 1.2 mg/L, Slope: 2.4

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: lower 95 % CL:1.2 mg/L, upper CL: 2.0 mg/L, Slope: 2.4

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

ca. 7.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

ca. 3.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

ca. 0.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

ca. 0.24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: One daphnia was trapped at the water surface in test medium (0.49 mg/L) after 24 and 48 hours of exposure. No other signs of disease or stress were observed.
- Observations on body length and weight: not observed.
- Other biological observations: none.
- Mortality of control: none.
- Other adverse effects control: none.
- Abnormal responses: none.
- Any observations that might cause a difference between measured and nominal values: none. The test and control media were clear during the whole exposure time. The test media containing the two highest test substance concentrations were coloured light yellow throughout the exposure period. The remaining test and control media were colourless throughout the test period.

Results with reference substance (positive control):

To check the reliability of the test conditions for acute toxicity studies to Daphnia magna at Seibersdorf Labor GmbH a static reference test with potassium dichromate is conducted twice a year. The last reference test was conducted in July 2010 and gave a 24 h EC50 of 1.0 mg potassium dichromate per litre (95% confidence interval of 0.82 to 1.3 mg/L) which is within the acceptability range of 0.6 - 2.1 mg/L given by the OECD guideline 202.

Reported statistics and error estimates:

The EC50-values and their confidence limits were calculated with the moving average method of W. Thompson, modified by C.S. Weil, Drug and Chemical Toxicology, 6 (6), 595-603 (1983).

Table 1: Actual test substance concentrations:

 

Group	Nominal test substance concentration	Actual test substance concentration (mg/L) at		Geometric mean of the actual test substance concentration (mean actual) 0-48h	Difference (0-48 h) between the actual concentrations
	(mg/L)	0 h	48 h	(mg/L)	(%)
K (control)	0.00	<QL	<QL	<QL
C1	0.13	0.09	0.12	0.10	33 33
C2	0.25	0.25	0.23	0.24	-8.00
C3	0.50	0.52	0.46	0.49	-12.50
C4	1.00	0.98	0.76	0.86	-22.45
C5	2.00	1.89	1.83	1.86	-3.44
C6	4.00	3.80	2.53	3.10	-33.42
C7	8.00	8.05	7.22	7.62	-10.31

 

QL: Quantitation limit of the analytical method used (0.017 mg/L)

 

Table 2: Immobilisation

 

Group	Mean actual test substance concentration (mg/L)	Immobilisation at 24 h	Immobilisation at 48 h
K (Control)	<QL	0 / 20	0 / 20
C1	0.10	1 / 20	1 / 20
C2	0.24	0 / 20	0 / 20
C3	0.49	1 / 20	3 / 20
C4	0.86	4 / 20	8 / 20
C5	1.86	11 / 20	18 / 20
C6	3.10	18 / 20	20 / 20
C7	7.62	20 / 20	20 / 20

 

QL: Quantitation limit of the analytical method used (0.017 mg/L)

 

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (48h) of the test item to Daphnia magna was determined to be 0.91 mg/L in a static test according to OECD 202 (1992) under GLP conditions.

Executive summary:

The objective of this study was to determine the acute toxicity of the test material to Daphnia magna according to OECD 202 (1984) under GLP conditions. For this purpose, young Daphnia magna were exposed under static conditions in bottle system without pH adjustment of the test media. The Daphnia were exposed to a range of concentrations, nominally 0 (control), 0.13, 0.25, 0.50, 1, 2, 4 and 8 mg/L of the test substance dissolved in reconstituted water. Samples of the test media for determination of the actual test substance concentrations were taken immediately before introduction of the Daphnia and after 48 hours. The measured concentrations were determined to be 0.10, 0.24, 0.49, 0.86, 1.86, 3.10 and 7.62 mg/L. The samples were analyzed using HPLC with UV-detection and external calibration for quantification. Measured concentrations ranged from 72 – 104 % of nominal values. For the confirmation of the sensitivity of the test animals of the used batch the results of a reference test with potassium dichromate were provided. After 24 hours the EC50 of potassium dichromate was determined to be 1.26 mg/L. The validity criteria of the test were fulfilled. Observations were made on the swimming ability and the immobilisation rate, respectively, after 24 and 48 hours of exposure. During the test a temperature range of 20 – 22 °C was maintained in the test vessels. Under the given conditions of the study the 48 h EC50 value for Daphnia was determined to be 0.91 mg/L.

Description of key information

The EC50 (48h) of the test item to Daphnia magna was determined to be 0.91 mg/L in a static test (reference 6.1.3-1) according to OECD 202 (1992) under GLP conditions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.91 mg/L

Additional information

The objective of this study was to determine the acute toxicity of the test material to Daphnia magna according to OECD 202 (1984) under GLP conditions. For this purpose, young Daphnia magna were exposed under static conditions in bottle system without pH adjustment of the test media. The Daphnia were exposed to a range of concentrations, nominally 0 (control), 0.13, 0.25, 0.50, 1, 2, 4 and 8 mg/L of the test substance dissolved in reconstituted water. Samples of the test media for determination of the actual test substance concentrations were taken immediately before introduction of the Daphnia and after 48 hours. The measured concentrations were determined to be 0.10, 0.24, 0.49, 0.86, 1.86, 3.10 and 7.62 mg/L. The samples were analyzed using HPLC with UV-detection and external calibration for quantification. Measured concentrations ranged from 72 – 104 % of nominal values. For the confirmation of the sensitivity of the test animals of the used batch the results of a reference test with potassium dichromate were provided. After 24 hours the EC50 of potassium dichromate was determined to be 1.26 mg/L. The validity criteria of the test were fulfilled. Observations were made on the swimming ability and the immobilisation rate, respectively, after 24 and 48 hours of exposure. During the test a temperature range of 20 – 22 °C was maintained in the test vessels. Under the given conditions of the study the 48 h EC50 value for Daphnia was determined to be 0.91 mg/L.