Dibenzoyl-L-tartaric acid monohydrate

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:

31/07/2003 - 04/11/2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 12.5 and 100 mg/L
- Sampling method: At the start (after 0 h) and at the end of the test (after 72 h) the inoculated replicates were analysed.
- Sample storage conditions before analysis: none (analysed immediately)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was stirred in test medium for about 20 min at approximately 20 ± 2ºC. The nominal concentration of the stock solution was 111 mg/I .
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no. The stock solution was solved and clear.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae - Chlorophytes
- Strain: CHODAT SAG 86.81
- Source: SAG (Collection of algal cultures in Göttingen)
- Method of cultivation: a seed culture was incubated for 4 days at 23 ± 2 ºC; the seed-culture was taken to inoculate a pre-culture (initial cell density: 1 E4 cells/ml). The pre-culture was incubated for 3 days at 23 ± 2 ºC. Final cell density: 122 E4 cells/ml.

ACCLIMATION
- Acclimation period: 3
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 2 ºC

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 ml) plugged with gas permeable siliconsponge caps
- Material, size, headspace, fill volume: glass, 250 ml, 150 ml, 100 ml.
- Initial cells density: 1 E4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- OECD medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: permanent illumination
- Light intensity and quality: Artificial light, type universal white (OSRAM L25), about 60 - 120 μE/(m2·s) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber (Neubauer improved) after 72h; fluoresence after 0, 24, 48 and 72h.
- Chlorophyll measurement: in vivo chlorophyll-a-fluorescence (pulsed excitation with light flashes having a wavelength of 435 nm)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes (no further data).
- Test concentrations: 6.25 - 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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

Duration:

72 h

Dose descriptor:

EC90

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.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

87.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The 72h-EC50 of the positive control was 0.47 mg/l (last control 18/02/2003, project 02/0106/60/11, non-GLP)

Table 1. pH values in the test solutions (pa = parallel)

Conc. (mg/l)	pH value
	Uninoculated (pa.5) 0 h	Uninoculated (pa.6) 72 h	Uninoculated (pa.1) 72 h
Control	8.2	8.3	8.7
6.25	6.2	7.5	6.4
12.5	7.0	8.2	8.3
25	7.5	8.2	8.4
50	7.7	8.2	8.5
100	7.9	8.3	8.5

 

Table 2. Measured fluorescence (relative units) after 0 h, 24 h, 48 h and 72 h (no = not inoculated).

Time (h)	Flask No.	Concentration (mg/l)
		0 (control)	100	50	25	12.5	6.25
0	1	30	28	27	29	28	29
	2	29	28	29	28	30	29
	3	33	29	28	30	28	29
	no	0	0	0	0	0	0
24	1	160	150	150	150	150	160
	2	150	150	160	150	150	160
	3	180	140	160	160	160	160
	no	0	0	0	0	0	0
48	1	890	690	880	860	820	790
	2	780	680	920	850	810	800
	3	1010	700	870	810	810	840
	no	0	0	0	0	0	0
72	1	3290	520	3070	3060	3250	2730
	2	2840	520	2910	3160	2890	2780
	3	3540	470	2850	3030	2930	2940
	no	0	0	0	0	0	0

 

Table 3. Cell density after 0 h, 24 h, 48 h and 72 h (no = not inoculated).

Time (h)	Flask No.	Concentration (mg/l)
		0 (control)	100	50	25	12.5	6.25
0	1	1.8	1.7	1,6	1,7	1,7	1,7
	2	1.7	1.7	1.7	1.7	1.8	1.7
	3	2.0	1.7	1.7	1.8	1.7	1.7
	mean	1.8	1.7	1.7	1.7	1.7	1.7
24	1	9.5	8.9	8.9	8.9	8.9	9.5
	2	8.9	8.9	9.5	8.9	8.9	9.5
	3	10.7	8.3	9.5	9.5	9.5	9.5
	mean	9.7	8.7	9.3	9.1	9.1	9.5
48	1	52.9	41.0	52.3	51.1	48.7	46.9
	2	46.3	40.4	54.6	50.5	48.1	47.5
	3	60.0	41.6	51.7	48.1	48.1	49.9
	mean	53.1	41.0	52.9	49.9	48.3	48.1
72	1	195.4	30.9	182.3	181.8	193.0	162.2
	2	168.7	30.9	172.8	187.7	171.7	165.1
	3	210.3	27.9	169.3	180.0	174.0	174.6
	mean	191.5	29.9	174.8	183.1	179.6	167.3

 

 

Table 4. Calculation of biomass and inhibition of biomass after 72 h.

Time (h)	Flask No.	Concentration (mg/l)
		0 (control)	100	50	25	12.5	6.25
Biomass (rel)	1	156	61	148	147	150	133
	2	135	61	146	149	138	135
	3	171	60	142	143	140	142
	mean	154	60	145	146	143	137
Inhibition (%)		0.0	60.7	5.5	5.0	7.1	11.0

 

Table 5. Calculation of growth rate and inhibition of growth rate after 72 h.

Time (h)	Flask No.	Concentration (mg/l)
		0 (control)	100	50	25	12.5	6.25
Biomass (rel)	1	0.0652	0.0406	0.0657	0.0647	0.0660	0.0631
	2	0.0637	0.0406	0.0640	0.0656	0.0634	0.0634
	3	0.0649	0.0387	0.0642	0.0641	0.0646	0.0642
	mean	0.0646	0.0399	0.0647	0.0648	0.0647	0.0635
Inhibition (%)		0.0	38.2	-0.1	-0.3	-0.1	1.7

Validity criteria fulfilled:

yes

Remarks:

The 72h-EC50 of the positive control was 0.47 mg/l (last control 18/02/2003, project 02/0106/60/11, non-GLP); the cell multiplication factor in the untreated control was 114-fold after 72h; the pH variation was no more than 2 units in the control.

Conclusions:

Under test conditions, the 72h-EC50 of the test item in freshwater algae is > 100 mg/L (nominal, based on growth rate). The NOEC is 50 mg/L and the LOEC 100 mg/L (nominal).

Executive summary:

A freshwater algae growth inhibition test was performed on the test item, according to OECD 201 / EU Method C.3, under GLP conditions. Based on the results of a range finding study, 3 replicate cultures of Desmodesmus subspicatus per concentration were exposed to 0 (negative control), 6.25, 12.5, 100 mg/L test item (nominal) in OECD medium for 72h under static conditions. Test concentrations were analytically determined by HPLC at the beginning and at the end of the test and did not deviate more than 20%. The results of the last performed positive control (potassium dichromate) were used to ensure the quality of the results. All validity criteria were met. Under test conditions, the 72h-EC50 of the test item in freshwater algae was determined to be > 100 mg/L (nominal, basis for effect: growth rate). The overall NOEC was determined to be 50 mg/L (nominal) and the overall LOEC was determined to be 100 mg/L (nominal).

Description of key information

Key stuy. Method according to OECD 201 / EU C.3, GLP study. Under test conditions, the 72h-EC50 of the test item in freshwater algae is > 100 mg/L (nominal, based on growth rate). The NOEC is 50 mg/L and the LOEC 100 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L