Succinic anhydride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April 2010 - 04 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: DSM Fine chemicals; No. LPBS2A2005
- Expiration date of the lot/batch: None
- Purity: Ca. 99.7 %
- Purity test date: 11-02-2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: Stable
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Stable

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

Vehicle:

no

Details on test solutions:

First experiment - A stock solution with a nominal test substance concentration of 111 mg/L was prepared by dissolving 111.4 mg test substance in 1 L of nutrient medium by stirring 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 111.2 mg test substance in 1 L of nutrient medium by manual homegenisation. The pH of the stock solution (4.05) was adjusted with 0.1 M NaOH to the pH of nutrient medium (7.46). Afterwards the stock solution was filtrated (0.2 µm, Minisart, Sartorius) to maintain sterile conditions.

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 am Rhein, Germany.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature was stable throughout the test periods (22–23 °C) in both of the experiments.

pH:

First experiment: The pH was between 4.2 and 7.5 at the start of the incubation in the test cultures and it was 7.4 in the control cultures. After 72 hours of incubation the pH was between 4.3 and 7.6 in the test cultures and it was 7.4 in the control cultures.Second experiment: The pH was between 7.3 and 7.5 at the start of the incubation, the pH was 7.3 in the control cultures. At the end of the test the pH was between 8.4 and 9.5 in the test cultures and it was 9.2 in the control cultures.

Nominal and measured concentrations:

First experiment, nominal concentrations: Control, 0.39, 0.78, 1.56, 3.13, 6.30, 12.5, 25, 50 and 100 mg/L. The limit of quantification of the analytical method was 3.55 mg/L, therefore the four lowest nominal concentrations could not be analysed. Geometric mean measured concentrations in the remaining test media were 6.20, 13.96, 23.5, 50.41 and 96.37 mg/L.
Second experiment, nominal concentrations: Control, 0.39, 0.78, 1.56, 3.13, 6.30, 12.5, 25, 50 and 100 mg/L. The limit of quantification of the analytical method was 3.55 mg/L, therefore the four lowest nominal concentrations could not be analysed. Geometric mean measured concentrations in the remaining test media were 4.47, 11.9, 24.02, 45.96 and 110.47 mg/L.

Details on test conditions:

At the onset of the experiments the cell concentration in the pre-culture was determined and the inoculum was prepared by diluting an appropriate volume of the pre-culture with nutrient medium to give a calculated cell concentration of 10exp5 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 (10exp5 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. In the first experiment the light intensity was between 5250 and 5980 lux with a maximum deviation from the mean light intensity of 8 %. In the second experiment the light intensity was between 6040 and 6960 lux with a maximum deviation of 7 %. 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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

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 period of the experiments.Microscopic evaluation of the inoculum at the start of the test revealed normal appearance of the algae in both experiments. At the end of the test morphological changes of the algae (disruption of cell membranes) were observed in the group containing the highest test substance concentration in the first experiment. All other test cultures showed normal appearance of the algae at the end of the test.

Results with reference substance (positive control):

The last reference test with K2Cr2O7 was conducted from the 21st to the 24th of June 2010 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg K2Cr2O7/L, respectively. These results establish the reliability of the test procedures for this kind of 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).

 

Table 1: 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	<QL	<QL	-
0.78	<QL	<QL	-
1.56	<QL	<QL	-
3.13	<QL	<QL	-
6.30	6.77	5.36	6.02
12.5	13.88	14.05	13.96
25	25.18	21.93	23.50
50	50.75	50.07	50.41
100	99.08	93.73	96.37
100 (blank, no algae)	99.30	98.22	98.76
Second experiment
Control	<QL	<QL	-
0.39	<QL	<QL	-
0.78	<QL	<QL	-
1.56	<QL	<QL	-
3.13	<QL	<QL	-
6.30	4.02	4.98	4.47
12.50	12.56	11.28	11.90
25.0	23.64	24.41	24.02
50.0	47.94	44.06	45.96
100	123.11	99.12	110.47
100 (blank, no algae)	97.87	88.98	93.32

QL: Quantification limit of the analytical method used (355 mg/L)

 

Table 2: 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	763.2	167.9	1.44	0.07	-	-
0.39	1066.3	425.5	1.54	0.13	-39.9	-6.9
0.78	1032.4	220.5	1.54	0.07	-35.4	-7.0
1.56	692.1	275.6	1.40	0.15	9.2	3.1
3.13	727.2	232.5	1.42	0.12	4.6	1.5
6.3	883.8	85.3	1.50	0.03	-15.9	-3.8
12.5	950.0	439.7	1.50	0.15	-24.6	-4.0
25	1048.6	252.4	1.55	0.08	-37.6	-7.3
50	89.9	79.1	0.65	0.38	88.2	54.7
100	-0.8	6.8	-0.15	0.42	100.1	110.5
Second experiment
Control	1087.5	86.0	1.57	0.03	-	-
0.39	1224.9	145.9	1.60	0.04	-12.6	-2.5
0.78	1328.4	175.4	1.63	0.04	-22.2	-4.2
1.56	1065.3	63.2	1.56	0.02	2.0	0.4
3.13	1188.2	80.6	1.59	0.02	-9.3	-1.9
6.3	1015.7	128.5	1.54	0.04	6.6	1.5
12.5	1109.0	218.4	1.57	0.07	-2.0	-0.2
25	938.6	97.4	1.52	0.04	13.7	3.1
50	1231.5	268.9	1.60	0.08	-13.2	-2.3
100	1234.7	184.6	1.61	0.05	-13.5	-2.6

Negative figures for inhibition indicate algal growth.

Validity criteria fulfilled:

yes

Conclusions:

Algae were exposed over a 72 hour period to succinic 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. Based on the nominal exposure concentration both the 72-hour EyC50 (cell density/biomass) and ErC50 (growth rate) were determined to be >100 mg/L with corresponding NOECs of 100 mg/L. 

Executive summary:

In a 72 hour acute toxicity study, cultures of Pseudokirchneriella subcapitata were exposed to succinic 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 under static conditions in accordance with the OECD guideline 201 (Version 23 March 2006). A first experiment without pH correction and second experiment with pH correction were conducted. The limit of quantification for succinic acid was 3.55 mg/L, therefore the four lowest concentrations were not quantified. Geometric mean measured concentrations of the remaining test vessels were 6.02, 13.96, 23.50, 50.41, 96.37, 98.76 mg/L in the first experiment and 4.47, 11.9, 24.02, 45.96 and 110.47 mg/L in the second experiment. This test was conducted twice as effects were seen in the first experiment likely originating from pH change (4.2–7.5 in the first experiment, compared to 7.4–7.5 in the second). In the first experiment, the NOEC and EC₅₀ values based on cell density were 25 and 40.7 mg/L, respectively. The NOEC and EC₅₀ values based on growth rate were 25 and 46.8 mg/L, respectively.  In the second test, the NOEC and EC₅₀ values based on cell density and growth rate were 100mg/L and > 100 mg/L, respectively.   

The following abnormalities were noted in the second experiment:  at the end of the test morphological changes of the algae (disruption of cell membranes) were observed in the group containing the highest test substance concentration in the first experiment. All other test cultures showed normal appearance of the algae at the end of the test. It is unclear if these results are from the first or second study.  

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 succinic acid toxicity is the 72 hour EC₅₀value of > 100 mg/L obtained in the experiment with pH adjustment. 

This toxicity study is classified as acceptable and satisfies the guideline requirements for toxicity to aquatic algae and cyanobacteria toxicity study.  

Results synopsis 

Test organism: Pseudokirchneriella subcapitata 

Test type: Static                     

72 hr EC₅₀: > 100 mg/L (biomass and growth rate, respectively)          

72 hr NOEC: 100 mg/L and > 100 mg/L  (biomass and growth rate, respectively)                   

Endpoint(s) effected: Biomass, growth rate

Description of key information

The potential toxicity of succinic anhydride to aquatic algae is assessed via read-across from its hydrolytic transformation product succinic acid.

In a 72 hour acute toxicity study, cultures of Pseudokirchneriella subcapitata were exposed to succinic 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 under static conditions in accordance with the OECD guideline 201. Geometric mean measured concentrations of the remaining test media were 6.02, 13.96, 23.50, 50.41, 96.37, 98.76 mg/L in the first experiment and 4.47, 11.9, 24.02, 45.96 and 110.47 mg/L in the second experiment. This test was conducted twice as effects were seen in the first experiment originating from pH change (4.2–7.5 in the first study, compared to 7.4–7.5 in the second).   

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 succinic anhydride toxicity is the 72 hour EC₅₀ value of > 100 mg/L corresponding to 85 mg/L succinic anhydride obtained in the study with pH adjustment.

Key value for chemical safety assessment

EC50 for freshwater algae:

85 mg/L

Additional information

At the end of the test morphological changes of the algae (disruption of cell membranes) were observed in the group containing the highest test substance concentration in the first experiment. All other test cultures showed normal appearance of the algae at the end of the test.