DL-malic acid

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

48 hours

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted according to OECD guideline 202 (2004)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fumaric acid

EC Number:

203-743-0

EC Name:

Fumaric acid

Cas Number:

110-17-8

IUPAC Name:

but-2-enedioic acid

Details on test material:

- Name of test material (as cited in study report): Fumaric acid
- Molecular formula (if other than submission substance): C4H4O4
- Smiles notation (if other than submission substance): C(=C/C(=O)O)\C(=O)O
- InChl (if other than submission substance): 1/C4H4O4/c5-3(6)1-2-4(7)8/h1-2H,(H,5,6)(H,7,8)/b2-1+
- Physical state: white powder
- Lot/batch No.: LPFS6A4011

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of the test media for determination of the actual test substance concentrations were taken immediately before introduction of the daphnia, before and after the change of the test medium at 24 hours and after 48 hours. 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 at 24 hours (new media). The pooled test media of the replicates were used for test substance determination at 24 hours (old media) and at the end of the test. One sample each was taken and then analysed in duplicate by HPLC. The samples were analysed at the day of sampling.

Test solutions

Details on test solutions:

For the first experiment stock solutions were prepared at the start of the test and 24 hours thereafter by addition of 25.0 mg (0 h) and 25.0 mg (24 h) test substance per 250 mL reconstituted water according to ISO 6341 and ultrasonication for 5 minutes. The test media were prepared by diluting the stock solutions with reconstituted water.

Also for the second experiment stock solutions were prepared at the start of the test and 24 hours thereafter by addition of 25.9 mg (0 h) and 26.3 mg (24 h) test substance per 250 mL reconstituted water according to ISO 6341 and stirring for 25 minutes. The test media were prepared by diluting the stock solutions with reconstituted water.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Neonates of Daphnia magna (Crustacea, Cladocera), less than 24 hours old, were obtained by hatching ephippia, supplied in the microbiotest "Daphtoxkit F TM " (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM).

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 investigation at approximately 88.5 hours after initiation of hatching in the first experiment and 85 hours after hatching in the second experiment. Approximately 2 hours 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.

Batch No. of the ephippia used: DM161209 (for both experiments).

Study design

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

260 mg CaCO3/L (first experiment), 230 mg CaCO3/L (second experiment)

Test temperature:

19-21°C (first experiment), 20-22°C (second experiment),

pH:

Due to the acid nature of the test substance the pH of the relevant test media ranged from 3.7 (100 mg/L) to 7.6 (6.25 mg/L). The pH of the control medium was 7.6.
The pH in the second experiment was between 7.3 and 8.0 in the 100 mg/L test medium and between 7.6 and 8.3 in the control media.

Dissolved oxygen:

8.5 - 8.8 mg O2/L (first experiment), 8.3 - 8.5 mg O2/L (second experiment)

Salinity:

Not applicable

Nominal and measured concentrations:

Experiment 1 - nominal concs - control, 6.25, 12.5, 25, 50 and 100 mg/L - measured concs - 6.02, 12.15, 23.09, 47.5 and 98.90 mg/L
Experiment 2 - nominal concs - control and 100 mg/L - measured conc - 95.95 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

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 K2Cr2O7 is conducted at least yearly. The last reference test was conducted in December 2009 and gave a 24 h EC50 of 1.1 mg K2Cr2O7 per litre (95% confidence interval of 0.86 to 1.5 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)

Any other information on results incl. tables

Mean measured concentrations in the two experiments

Nominal concentration (mg/L)	Measured concentration (mg/L)				Mean (mg/L)
	0 hours (new)	24 hours (old)	24 hours (new)	48 hours (old)
First experiment
Control	<QL	<QL	<QL	<QL	-
6.25	6.21	6.09	6.36	5.44	6.02
12.5	12.36	12.2	12.58	11.49	12.15
25	23.5	22.84	23.76	22.29	23.09
50	47.76	47.0	48.42	46.93	47.52
100	100.24	98.4	98.32	98.65	98.90
Second experiment
Control	<QL	<QL	<QL	<QL	-
100	98.16	96.92	95.34	93.42	05.95

QL: Quantification Limit of the analytical method used (0.16 g/L)

 

 

Immobilised Daphnia in the two experiments

Nominal concentration (mg/L)	Immobility
	24 hours		48 hours
	number	%	number	%
First experiment
Control	0	0	0	0
6.25	0	0	0	0
12.5	1	5	1	5
25	2	10	2	10
50	2	10	2	10
100	20	100	20	100
Second experiment
Control	1	5	1	5
100	0	0	1	5

20Daphniawere exposed in each treatment

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the fact that the effects seen in the first experiment were influenced by the pH change the most appropriate endpoint to use for the assessment of fumaric toxicity is the 48 hour EC50value of >100 mg/L obtained in the study with pH adjustment. 

Executive summary:

Read across to fumaric acid is considered valid and malic acid is concluded to have low toxicity to aquatic organisms and classification is not required.

The rational for read across is that fumaric acid will metabolise in biological systems to malic acid. Fumaric acid is also slightly more fat soluble and is considered more likely to absorb to membranes and increase exposure to aquatic organisms.

The 48 hour acute toxicity of fumaric acid to Daphnia magna was determined in two semi-static tests according to the OECD 202 guideline.

In the first experiment daphnids were exposed over a 48 hour period to fumaric acid at nominal concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L. pH ranged from 3.7 in the 100 mg/L treatment to 7.4 in the 6.25 mg/L treatment in a dose related response.  Concentrations of fumaric acid were verified analytically at test initiation, after 24 hours in both new and old test media and test termination.  Mean measured concentrations was 6.02, 12.15, 23.09, 47.52 and 98.90 mg/L. The 48-hour EC₅₀was determined to be 66 mg/L (nominal) with 95% confidence limits of 59 to 73 mg/L.

In the second experiment daphnids were exposed over a 48 hour period to fumaric acid at a nominal concentration of 0 (control) and 100 mg/L. The pH of the 100 mg/L was 8.3 at the start of each exposure period.   The mean measured concentration of fumaric acid was 95.95 mg/ L. The 48-hour EC₅₀was determined to be >100 mg/L (nominal).

Based on the fact that the effects seen in the first experiment were influenced by the pH change the most appropriate endpoint to use for the assessment of fumaric toxicity is the 48 hour EC₅₀value of >100 mg/L obtained in the study with pH adjustment.