DL-malic acid

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

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

Adequacy of study:

key study

Study period:

3 hours

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted according to OECD guideline 209 (1984)

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 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

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:

no

Details on sampling:

Not applicable

Test solutions

Vehicle:

no

Details on test solutions:

Fumaric acid was not enough soluble in water to prepare a stock solution of nominal 1.5 g dry weight/L. Therefore a less concentrated solution was prepared by diluting 528.1 mg test substance in 1000 mL tap water. This stock solution was used for the preparation of the different incubation mixtures.

All incubation mixtures were prepared by adding 200 mL of microbial inoculum and 16 mL of concentrated synthetic sewage solution to 284 mL of tap water and/or the appropriate amount of the test or control substance in glass beakers with a nominal volume of 1000 mL.

After mixing, the samples were aerated at a flow rate of 0.7 L/min using oil free compressed air and a Pasteur-pipette as aeration device. The incubation of the individual samples was started at intervals of 12-13 min and each sample was aerated for 3 hours at 20 °C. At the end of the incubation time the pH was determined and aliquots of the samples were transferred to measuring bottles and oxygen consumption was recorded

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

For the preparation of the microbial inoculum activated sludge was used. The sludge was collected from one of the return sludge channels of the sewage treatment plant in Baden near Vienna (Zubringerstraße, A-2500 Baden). Baden is a spa with only a small amount of industry and the waste water of the sewage treatment plant is predominantly domestic.

A sample of activated sludge was collected on the day of the test. The container for transportation was only maximally 2/3 filled in order to maintain the contact with air. On arrival in the laboratory (less than 1 hour after sample collection), the supernatant over the sludge was decanted, the sludge was diluted with tap water and aerated by means of compressed air. The concentration of suspended solids was determined by filtering 3 mL of the sample through a pre-dried and pre-weighed glass microfibre filter. After drying at 110 °C and reweighing the amount of solids was calculated.

On the basis of this calculation the concentration of suspended solids was adjusted to 4 g/L with tap water.



The concentration of the microbial inoculum was adjusted to 4 g of dry weight per litre which results in a final concentration of 1.6 g dry weight per litre in the samples.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not applicable

Test temperature:

The temperature in the samples was 20 ± 2 °C.

pH:

The pH of the standardised activated sludge was 7.8. The pH of the incubation mixtures after the 3 hour incubation was between 8.1 and 8.4, the
pH of the negative control samples was 8.4 and 8.5.

Dissolved oxygen:

Respiration rates (in mg O2.L-1.h-1 ) were determined in aliquots of the incubation mixtures. Bottles with narrow neck and a volume of about 100 mL were filled with the sample and positioned on a magnetic stirrer. Then the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottle neck to avoid contact of the sample with the atmosphere. The decline of oxygen concentration was measured by recording the concentrations of oxygen at various times.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal exposure concentrations were 0 (control), 7.7, 19.2, 48, 120 and 300 mg/L

Details on test conditions:

Negative controls were run before the first and after the last test substance sample. After incubation the respiration rates were determined in closed bottles using an oxygen sensitive electrode. The inhibition of respiration was calculated from the respiration rates using the mean value of the negative controls as 100 %.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (Aldrich, Art. No. D 7,060-0, Lot 15809KI, purity according to the certificate of analysis: 99.7 %).

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L. The actual values of the EC20, EC50 and EC80 for 3,5-dichlorophenol were: EC20 = 8.4 mg/L EC50 = 18.3 mg/L EC80 = 40.1 mg/L

Reported statistics and error estimates:

The concentrations of the test substance around 50 % inhibition (if such data exist) and the concentrations of the positive control are converted to log10. These values and the corresponding percentage of inhibition are subjected to linear regression analysis. These analyses are used to obtain the regression coefficients and the respective EC20, EC50 and EC80 values.

Any other information on results incl. tables

Respiration rates and inhibition of samples

Sample	Conc. (mg/L)	mg O2/L at start	mg O2/L at end	Respiration rate (mg O2/L/h)	% inhibition
Control 1	0	8.06	6.31	12.21	5.33
Fumaric acid	7.7	7.78	6.24	12.35	4.21
	19	7.65	5.63	15.3	-18.66
	47.5	7.41	5.51	15.16	-17.55
	120.4	7.48	5.47	16.30	-26.37
	300	7.56	5.53	16.55	-28.32
Control 2	0	8.03	6.40	13.58	-5.33
3,5-DCP	5.0	7.65	6.10	12.92	-0.16
	12.3	8.60	7.64	8.28	35.83
	30.2	9.15	8.65	4.05	68.56

Negative values indicate enhanced respiration compared to the control cultures.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 3 -hour EC50 based on respiration inhibition was >300 mg fumaric acid/L.

Executive summary:

Activated sludge obtained from a sewage treatment plant was exposed in a 3 -hour respiration inhibition test to concentrations of 0 (control), 7.7, 19. 47.5, 102.4 and 300 mg fumaric acid/L. The test design included duplicate samples and followed OECD 209 guidelines. The 3 -hour EC50 based on respiration inhibition was >300 mg fumaric acid/L.