Myrac Aldehyde

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:

04 Aug 2017 - 21 Sep 2017

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:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, individually prepared WAFs at loading rates of 0.46, 1.0, 2.2, 4.6, and 10 mg/L
- Frequency: at start of test and after 24 h from the freshly prepared solutions, and at the first renewal (24 h) and the end of the test from the 24h-old solutions.
- Sampling method: 4.0 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis:Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of each refreshment period, the replicates were not pooled at each concentration before sampling. Instead, samples were taken from the same replicate at each test concentration

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with loading rates individually prepared at 0.46, 1.0, 2.2, 4.6 and 10 mg/L. A 3-day period of magnetic stirring was applied to ensure homogenisation and maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. The entire preparation of the WAFs occurred in completely closed vessels with very limited headspace.
- Controls: Test medium without test item or other additives.
- All test solutions were clear and colourless at the end of the preparation procedure.

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 test organisms at start of test: <24h old
- Feeding during test: no feeding
- 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.

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel
- Maximum age of cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of the medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae
- Medium: M7 medium.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20-21 °C

pH:

Start: 7.6-7.8
24 h (fresh): 7.6-7.7
48 h (old): 7.7-7.8

Dissolved oxygen:

Start: 8.3-8.8
24 h (fresh): 8.3-8.8
48 h (old): 8.5-8.8

Nominal and measured concentrations:

Nominal loading rates: WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Measured concentrations: See Table 1 in "Any other information on results incl. tables".
Average exposure concentrations: 0.035, 0.10, 0.70, 2.5 and 3.9 mg/L (for calculation method please refer to section 'any other information on materials and methods')

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL all-glass, airtight closed with no headspace
- Aeration: no
- Renewal rate of test solution: Semi-static with renewal of test solutions after 24 h.
- 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 with tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes (M7 medium used as culture medium)
- Intervals of water quality measurement:
* pH and dissolved oxygen: at the beginning of the test, after 24 h exposure (measured in freshly prepared solutions) and at the end of the test
* Temperature: Continuously in a temperature control vessel, beginning at the start of the test. Additionally, after 24 hours for all concentrations and the control, both in old and fresh medium.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h photoperiod daily

EFFECT PARAMETERS MEASURED
- Immobility (incl mortality): at 24 h and at 48 h.

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: control and WAF prepared at loading rates of 1.0 and 10 mg/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (reference test performed June 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval 0.31-0.60 mg/L

Details on results:

- Behavioural abnormalities: A number of Daphnia were observed trapped at the surface of the test solutions (see Table 2 in 'Any other information on results incl. tables'. These organisms were reimmersed into the respective solutions before recording of mobility.
- Immobility of control: 5% immobility at 24 and 48 h.
- The analytical results obtained in the final test did not show a relationship between the increasing loading rates and the measured concentrations at the start of the test. This was, however, not observed for the freshly prepared solutions at 24 hours of exposure. The reason for the discrepancy is unclear, since preparation of the WAFs was identical. The consequence is the apparent lack of a dose-response relationship after the first 24 hours of exposure when the observed effects are related to the average exposure concentrations. However, when immobility is related to the actual exposure concentrations, a clear dose-response is seen, since 0, 20, 5, 40 and 95% immobility was observed at initially measured concentrations of 0.045, 0.085, 0.71, 1.2 and 2.6 mg/L, respectively.
Additionally, 40% immobility was recorded at an average exposure concentration of 0.035 mg/L at the end of the final test. This was not in line with what was observed at higher exposure concentrations, since no immobility was observed at average exposure concentrations of 0.09 mg/L in the combined limit/range-finding test and at an average exposure concentration of 0.1 mg/L in the final test. Consequently, it was decided to exclude the lowest test concentration from calculation of the effects parameters.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. 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.
- Relevant effect levels:
* The 24h-EC50 was 0.95 mg/L with a 95% confidence interval between 0.85 and 1.1 mg/L.
* The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.66 mg/L.

Reported statistics and error estimates:

The 24 and 48h-EC50-values were calculated using the Spearman-Karber method (without trim) on the percentages of affected daphnids and the logarithms of the corresponding test item concentrations. The responses recorded at the lowest test concentration were excluded from the calculation.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1 Average Exposure Concentration Versus Nominal Concentration

 

MYRAC ALD BHT, WAF (mg/L)	Measured concentrations (mg/L)				Average exposure (mg/L)
	t=0h (fresh)	t=24h (old)	t=24h (fresh)	t=48h (old)
0.46	0.085	0.017	0.059	0.018	0.035
1.0	0.045	0.018	0.27	0.12	0.10
2.2	0.71	0.36	0.98	0.80	0.70
4.6	2.6	1.9	3.1	2.5	2.5
10	1.2	0.72	7.1	6.8	3.9

 

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

Time (h)	Replicate	Average exposure MYRAC ALD BHT (mg/L)
		Control	0.035	0.10	0.70	2.5	3.9
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	1	2	0	1	5^#	1
	B	0	1(1)	0	0	5(1)	2
	C	0	0	0	0	5	2
	D	0	1	0	0(2)	4	3
	Total immobilised	1	4	0	1	19	8
	Effect %	5	20	0	5	95	40
48	A	1	2	0	3	5	5
	B	0	4	0	3	5	5
	C	0	1(2)	0	3	5	5
	D	0	1*	0	5	5	5
	Total immobilised	1	8	0	14	20	20
	Effect %	5	40	0	70	100	100

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

* Considering the worst case scenario, one missing daphnia was scored as immobile.

^#Microscopic observation revealed no test item attached to the daphnids.

Table 3: Effect Parameters

Parameter	MYRAC ALD BHT Average exposure (mg/L)	95%-confidence interval (mg/L)
24h-EC50	2.1	1.6– 2.8
48h-EC50	0.43	0.31 – 0.60

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 3-CYCLOHEXENE-1-CARBOXALDEHYDE MULTICONSTITUENT was 0.43 mg/L (95% confidence interval between 0.31 and 0.60 mg/L).

Executive summary:

The acute toxicity to aquatic invertebrates was examined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study, groups of Daphnia magna (4 replicates of 5) were exposed to WAFs individually prepared at loading rates of o (control), 0.46, 1.0, 2.2, 4.6 and 10 mg/L for 48 hours under semi-static conditions. Nominal target concentrations were analytically verified at the start and the end of each renewal period and average test substance concentrations calculated as mathematical means of the concentrations at the start and the end of each renewal period. Average exposure concentrations were determined to be 0.035, 0.10, 0.70, 2.5 and 3.9 mg/L, and effect concentrations expressed as such. Immobility of daphnids was recorded after 24 and 48 hours exposure. The test is valid. After 48 hours exposure, daphnia immobility in the control was 5%. In the increasing test concentrations, immobility was reported to be 40, 0, 70, 100, and 100%. The 40% immobility recorded at an average exposure concentration of 0.035 mg/L was not in line with what was observed at higher exposure concentrations, since no immobility was observed at average exposure concentrations of 0.09 mg/L in the combined limit/range-finding test and at an average exposure concentration of 0.1 mg/L in the final test. Consequently, it was decided to exclude the lowest test concentration from calculation of the effects parameters. Based on these findings, the 48-h EC50 value was determined to be 0.43 mg/L (95% C.I. 0.31-0.60 mg/L).

Description of key information

The acute toxicity to aquatic invertebrates was examined in a study according to OECD TG 202 and in compliance with GLP criteria. In this study, groups of Daphnia magna (4 replicates of 5) were exposed to WAFs individually prepared at loading rates of o (control), 0.46, 1.0, 2.2, 4.6 and 10 mg/L for 48 hours under semi-static conditions. Nominal target concentrations were analytically verified at the start and the end of each renewal period and average test substance concentrations calculated as mathematical means of the concentrations at the start and the end of each renewal period. Average exposure concentrations were determined to be 0.035, 0.10, 0.70, 2.5 and 3.9 mg/L, and effect concentrations expressed as such. Immobility of daphnids was recorded after 24 and 48 hours exposure. The test is valid. After 48 hours exposure, daphnia immobility in the control was 5%. In the increasing test concentrations, immobility was reported to be 40, 0, 70, 100, and 100%. The 40% immobility recorded at an average exposure concentration of 0.035 mg/L was not in line with what was observed at higher exposure concentrations, since no immobility was observed at average exposure concentrations of 0.09 mg/L in the combined limit/range-finding test and at an average exposure concentration of 0.1 mg/L in the final test. Consequently, it was decided to exclude the lowest test concentration from calculation of the effects parameters. Based on these findings, the 48-h EC50 value was determined to be 0.43 mg/L (95% C.I. 0.31-0.60 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.43 mg/L