Tripotassium hexacyanoferrate

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:

24 Apr 2018 - 04 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other 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:

- Concentrations: Control and all test concentrations
- Sampling method: 3.0 mL taken from the approximate centre of additional vessels without Daphnia, at t=0 h and t=48 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to test medium. Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions in the final test were light yellow to yellow at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: >2 weeks old
- Age at test initiation: <24 hours
- Validity of batch: 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.

CULTIVATION
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L (as CaCO3)

Test temperature:

20-22°C

pH:

At start: 8.0 - 8.1
At 48 h: 8.1 - 8.2

Dissolved oxygen:

At start: 8.5 - 8.9 mg/L
At 48 h: 9.0 - 9.2 mg/L

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56, 100, 180 mg/L
Measured concentrations: 10.6, 21.2, 28, 54.7, 102, 172 mg/L; 86-118% relative to nominal throughout the test. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations. See Table 1 in 'Any other information on results' for details on the measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL all-glass, containing 50 mL of test solution
- Aeration: no
- Feeding: 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: Adjusted ISO medium, prepared with tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes, M7 medium
- Intervals of water quality measurement: Dissolved oxygen and pH: at beginning and end of the test, for all concentrations and the control. Temperature: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: test was performed in the dark.

EFFECT PARAMETERS MEASURED
- Immobility (including mortality) at 24 hours and at 48 hours.

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: Control, 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected EC50 was > 100 mg/L (nominal concentration).

LIMIT TEST
- Test concentrations: Control, 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected EC50 was < 100 mg/L (nominal concentration).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed March 2018)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

59 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% c.i.: 52-67 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: 5% entrapment at surface was observed in the Control group. This was considered not biologically relevant.
- Mortality of control: none
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
- A small negative response at the wavelength of the test item was detected in the spectrum of the blank test samples. Similar responses were found in the blank QC samples. It was considered that it has no significant effect on the results of the test samples.
- Test samples with concentrations between 10 and 56 mg/L can be seen as reliable regardless of being below the LOQ of the validated analytical method. All other analytical parameters (calibration curve, stability of the system, accuracy and repeatability) were in criteria and therefore the results are accepted.

Results with reference substance (positive control):

- Results with reference substance valid?
yes
- The 48h-EC50 was 0.33 mg/L with a 95% confidence interval between 0.29 and 0.38 mg/L.
- The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Reported statistics and error estimates:

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

Table 1: Final Test: Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis (a)	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	27 Jun 2018	04 Jul 2018	0	< 0.8 (b)	n.a.
			10	11.8	118
			18	19.1	106
			32	32.7	102
			56	63.8	114
			100	111	111
			180	173	96
48	29 Jun 2018	04 Jul 2018	0	< 0.8 (b)	n.a.	n.a.
			10	10.6	106	90
			18	21.2	118	111
			32	28 (c)	86	84
			56	54.7	98	86
			100	102	102	92
			180	172	95	99

(a)    Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(b)     A negative concentration was calculated, because the absorption was below the intercept of the calibration curve. Therefore the analyzed concentration is reported as < the lowest calibration solution (i.e. 0.8 mg/L).

^(c)      Estimated value, calculated by extrapolation of the calibration curve.

n.a. Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)	Replicate	Potassium Ferricyanide Nominal conc. (mg/L)
		Control	10	18	32	56	100	180
0	A	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20
24	A	0 (1)	0 (1)	0	0	0	0	0 (1)
	B	0	0	0	0	0	0	1
	C	0	0	0	0	0	0	0
	D	0	0	0	0	0	0	2
	Total immobilised	0	0	0	0	0	0	3
	Effect %	0	0	0	0	0	0	15
48	A	0 (1)	0	0	0	1	5	5
	B	0	0	0	0	2	5	5
	C	0	0	0	0	1	4	5
	D	0	0	0	0	5	5	5
	Total immobilised	0	0	0	0	9	19	20
	Effect %	0	0	0	0	45	95	100

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:

yes

Remarks:

For details on validity criteria, please refer to section 'Overall remarks'

Conclusions:

The 48h-EC50 for Daphnia magna exposed to the test item was 59 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 52 and 67 mg/L).

Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 10, 18, 32, 56, 100 and 180 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). Measured concentrations were 86 -118% relative to nominal throughout the test, therefore effect parameters were based on nominal concentrations. The 48h-EC50 for Daphnia magna exposed to the test item was 59 mg/L (95% confidence interval: 52 - 67 mg/L). The study met all validity criteria and is considered to be reliable without restrictions.

Description of key information

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 10, 18, 32, 56, 100 and 180 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). Measured concentrations were 86 -118% relative to nominal throughout the test, therefore effect parameters were based on nominal concentrations. The 48h-EC50 for Daphnia magna exposed to the test item was 59 mg/L (95% confidence interval: 52 - 67 mg/L). The study met all validity criteria and is considered to be reliable without restrictions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

59 mg/L