DL-malic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

72 hours

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted according to OECD guideline 201 (2006)

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 201 (Alga, Growth Inhibition 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:

At the start and at the end of the incubation period, samples of the test media were drawn and the concentrations of the test substance were determined in aliquots
• of the blank containing test substance, but no algae;
• of one replicate of each of the test and control cultures (the 72 hours samples were filtered through a 0.20 µm filter)
One sample was taken of each of the above listed aliquots and immediately analysed in duplicate by HPLC

Test solutions

Details on test solutions:

First experiment - A stock solution with a nominal test substance concentration of 111 mg/L was prepared by dissolving 111.7 mg test substance in 1 L of nutrient medium by stirring for 20 minutes and subsequent ultrasonication for 5 minutes. Aliquots of this stock solution were diluted with nutrient medium to obtain lower concentrations.

Second experiment - A stock solution with a nominal test substance concentration of 111 mg/L was prepared by dissolving 112 mg test substance in 1 L of nutrient medium by stirring for 10 minutes. The pH of this solution (3.33) was adjusted with 0.1 M NaOH to 7.6 (according to the
physiological pH of the algal growth medium).

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) ATCC (American Type Culture Collection) 22662, obtained from LGC Promochem GmbH, Mercatorstraße 51, 46485 Wesel A Rhein, Germany.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not applicable

Test temperature:

The temperature was stable throughout the test periods (21-23 °C) in both of the experiments.

pH:

The pH in the first experiment was between 3.4 and 7.5 at the start of the incubation in the test cultures and it was 7.3 in the control cultures. After 72 hours of incubation the pH was between 3.5 and 9.2 in the test cultures and it was 7.3 in the control cultures.

The test culture in the second experiment had a pH value of 7.4 at the start of the experiment which remained constant during the exposure period. The control culture had a pH of 7.5 at the start of the incubation, which dropped to 7.2 after 72 hours of incubation.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

First experiment - nominal concs. - control, 0.39, 0.78, 1.56, 3.13, 6.30, 12.5, 25, 50 and 100 mg/L - geometric mean measured concs - 0.32, 0.74, 1.48, 3.21, 6.45, 10.95, 23.84, 49.94, 99.4 mg/L
Second experiment - nominal concs. - control and 100 mg/L - 89.07 mg/L.

Details on test conditions:

At the onset of the experiments the cell concentration in the preculture was determined and the inoculum was prepared by diluting an appropriate volume of the preculture with nutrient medium to give a calculated cell concentration of 10x5 cells/mL.
All test cultures and the blanks were set up in 250 mL conical glass flasks (Erlenmeyer). The total culture volume was 100 mL.
Each test substance culture consisted of 10 mL inoculum (105 algae/mL) and of 90 mL of the respective test substance preparation.
The blanks without algae contained 90 mL of the undiluted stock solution and 10 mL of nutrient medium.
The negative control cultures contained 10 mL of inoculum and 90 mL of nutrient medium.
All cultures and the blank were incubated at 21 - 24 (± 2) °C for three days in permanent light with an intensity between 4400 and 8800 lux while shaking.
For each experiment three replicate cultures were incubated for each test substance concentration and six replicates for the control.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No change in the colour of the media and no precipitation of test substance was noted during the incubation periods. Microscopic evaluation of the inoculum at the start of the tests and of the algae at the end of the tests revealed normal appearance of the algae.

Results with reference substance (positive control):

The last reference test with K2Cr2O7 was conducted from the 16th to the 19th of November 2009 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 0.87 and 0.65 mg K2Cr2O7/L, respectively, which reveals the reliability of the applied test procedure for the study type.

Reported statistics and error estimates:

The mean yield of the test cultures is expressed as percentage of the mean yield of the control cultures and from this the percentage inhibition in yield is calculated. The logarithms of the substance concentrations are then plotted against the corresponding percent inhibition. The EyC50 for the 72 h incubation period is taken from the intercept of this curve with the parallel drawn to the abscissa at 50 % inhibition.

Based on the yield as well as on the average growth rates two "lowest observed effective concentrations" (LOECs) are calculated for each experiment by comparison of the data of the three replicates of each test substance culture with the negative control (analysis of variance, followed by the Scheffé-test, P = 0.05). The "no observed effect concentrations" (NOECs) are then derived from these results (highest concentration with no statistically significant difference to the control).

Any other information on results incl. tables

Measured concentrations in the two experiments

Nominal concentration (mg/L)	Measured concentrations (mg/L)		Geometric mean (mg/L)
	0 hours	72 hours
First experiment
Control	<QL	<QL	-
0.39	0.33	0.31	0.32
0.78	0.73	0.76	0.74
1.56	1.51	1.46	1.48
3.13	3.35	3.07	3.21
6.25	6.56	6.36	6.45
12.5	11.08	10.82	10.95
25	23.99	23.69	23.84
50	50.38	49.51	49.94
100	99.69	99.11	99.40
100 (blank, no algae)	100.17	98.52	99,34
Second experiment
Control	<QL	<QL	-
100	93.7	84.63	89.06
100 (blank, no algae)	93.1	93.81	93.47

QL: Quantification limit of the analytical method used (0.055 mg/L)

Mean yield, average specific growth rates and percent inhibition

Nominal concentration (mg/L)	Yield (10000 cells/mL)  0 – 72 hours		Growth rate (per day) 0 – 72 hours		% inhibition
	Mean	SD	Mean	SD	Yield	Growth rate
First experiment
Control	1283.0	134.1	1.62	0.03	-	-
0.39	1190.2	152.2	1.59	0.04	7.2	1.6
0.78	1103.3	57.3	1.57	0.02	14.0	3.0
1.56	1317.9	222.8	1.63	0.05	-2.7	-0.4
3.13	1321.3	55.9	1.63	0.01	-3.0	-0.7
6.25	1242.9	60.4	1.61	0.02	3.1	0.6
12.5	1478.1	200.9	1.66	0.04	-15.2	-2.8
25	1450.4	33.7	1.66	0.01	-13.0	-2.6
50	22.3	20.9	0.32	0.21	98.3	80.1
100	1.0	5.3	-0.01	0.16	99.9	100.4
Second experiment
Control	1646.3	365.5	1.69	0.08	-	-
100	1557.7	296.9	1.68	0.07	5.4	0.9

Negative figures for inhibition indicate algal growth.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Algae were exposed over a 72 hour period to fumaric acid at a nominal concentration of 0 (control) and 100 mg/L. The pH of the 100 mg/L medium was 7.4 at the start of the exposure period.   The mean measured concentration of fumaric acid was 89.07 mg/ L. Based on the nominal exposure concentration both the 72-hour EyC50and ErC50were determined to be >100 mg/L with corresponding NOECs of 100 mg/L. 

Executive summary:

Two 72 hour algal inhibition experiments were conducted with fumaric acid and Pseudokirchneriella subcapitata according to the OECD 203 guideline.

In the first experiment algae were exposed over a 72 hour period to fumaric acid at nominal concentrations of 0 (control), 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L. pH ranged from 3.4 in the 100 mg/L treatment to 7.5 in the 0.39 mg/L treatment in a dose response relationship.  Concentrations of fumaric acid were verified analytically at test initiation and termination.  Geometric mean measured concentrations were 0.32, 0.74, 1.48, 3.21, 6.45, 10.95, 23.84, 49.94 and 99.40 mg/L. Based on nominal exposure concentrations the 72-hour E_yC₅₀was determined to be 37.2 mg/L with a corresponding NOEC of with 25 mg/L. The 72-hour E_rC₅₀was determined to be 38.9 mg/L with a corresponding NOEC of with 25 mg/L. 

In the second experiment algae were exposed over a 72 hour period to fumaric acid at a nominal concentration of 0 (control) and 100 mg/L. The pH of the 100 mg/L medium was 7.4 at the start of the exposure period.   The mean measured concentration of fumaric acid was 89.07 mg/ L. Based on the nominal exposure concentration both the 72-hour E_yC₅₀and E_rC₅₀were determined to be >100 mg/L with corresponding NOECs of 100 mg/L. 

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 72 hour E_rC₅₀value of >100 mg/L obtained in the study with pH adjustment.