L-arabinose

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:

2018-06-05 to 2018-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: AD16081001
- Expiration date of the batch: 2019-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Stable under ambient conditions

Analytical monitoring:

yes

Details on sampling:

- Concentrations: six replicate samples (2 mL per replicate) were taken from the test solution (100 mg/L) and from the control at the start and at the end of the test
- Sample storage conditions before analysis: No storage

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent.

Test organisms (species):

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

Details on test organisms:

- Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum)
- Strain Number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Origin: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding Conditions: The stock cultures are small algal cultures that are regularly planted on agar. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

In the test flasks: 22.6 – 23.1°C
In the climatic chamber: 22.1 - 23.9°C

pH:

7.72 – 9.01

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 100 mg/L
Mean measured: 35.62 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, fill volume: Glass, ~250 mL volume, 100 mL test medium
- Aeration: No
- No. of vessels per concentration: 6
- No. of vessels per control: 6
- Initial cell density: 10^4 cells/mL in each test flask

GROWTH MEDIUM
- Standard medium used: Yes, OECD Medium

TEST MEDIUM
- Preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water. Separate stock solutions were prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE Direct-Q 5 water purification system). Then, the growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light quality: Fluorescent lamps (400-700 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At 24, 48 and 72 h manual cell counting using microscope with counting chamber

STATISTICAL ANALYSIS
Mean values and standard deviations were calculated at the start, and on each subsequent day of the test using Microsoft Excel for Windows software. Statistical comparisons of average specific growth rates and yield in control and in treated group were not necessary. The EC values of the test item and the NOEC and LOEC values related to growth rates and yield were determined directly from the raw data.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

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:

EC50

Effect conc.:

> 35.62 mg/L

Nominal / measured:

meas. (geom. mean)

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:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 35.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

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.:

> 35.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 35.62 mg/L

Nominal / measured:

meas. (geom. mean)

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:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

35.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

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.:

35.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

VALIDITY OF THE TEST
Cell density in the control cultures increased by a factor of more than 16 (by a factor of 169.83) within 72 hours. This corresponds to a specific growth rate of 1.71 day^-1.

The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %.
- CV for section-by-section growth rate day 0-1: 6.40 %
- CV for section-by-section growth rate day 1-2: 8.91 %
- CV for section-by-section growth rate day 2-3: 4.56 %

The mean coefficient of variation for section-by-section specific growth rates: 6.63 %.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in test.
- CV for average specific growth rate day 0-3: 3.41 %

All validity criteria were met, therefore the study is considered as valid.

Results with reference substance (positive control):

72h ErC50: 1.08 mg/L, (95 % confidence limits: 0.79 – 1.51 mg/L)
72h EyC50: 0.61 mg/L, (95 % confidence limits: 0.49 – 0.76 mg/L)
The date of the last study with the reference item Potassium dichromate was: 26 February - 01 March 2018.

Reported statistics and error estimates:

As a limit test was performed the EC values of the test item and the NOEC and LOEC values related to growth rates and yield were determined directly from the raw data.

Table 1: Summary of biological endpoints

Endpoints (0-72 h)	Growth rate (r)	Yield (y)
EC10	> 100 mg/L (> 35.62 mg/L, measured)	> 100 mg/L (> 35.62 mg/L, measured)
EC20	> 100 mg/L (> 35.62 mg/L, measured)	> 100 mg/L (> 35.62 mg/L, measured)
EC50	> 100 mg/L (> 35.62 mg/L, measured)	> 100 mg/L (> 35.62 mg/L, measured)
NOEC	100 mg/L (35.62 mg/L, measured)	100 mg/L (35.62 mg/L, measured)
LOEC	> 100 mg/L (> 35.62 mg/L, measured)	> 100 mg/L (> 35.62 mg/L, measured)

Table 2: Algal cell number (x 10⁴ cell/mL) during the test period of 72 hours and Yield

Test Group		Number of Cells (× 10^4/mL)				Yield
		0 h	24 h	48 h	72 h
Control		1	4	28	168	167
		1	4	33	203	202
		1	5	27	161	160
		1	4	26	128	127
		1	4	27	156	155
		1	4	35	203	202
	Mean value	1.00	4.17	29.33	169.83	168.8
	SD	0.0	0.4	3.7	29.1	29.1
100 mg/L(nominal)		1	4	22	94	93
		1	4	32	187	186
		1	4	28	193	192
		1	4	30	206	205
		1	4	31	164	163
		1	4	28	153	152
	Mean value	1.00	4.00	28.50	166.17	165.2
	SD	0.0	0.0	3.6	40.3	40.3

Table 3: The specific growth rates during the test period of 72 hours

Test Group		Growth Rates (r)
		0 – 24 h	0 – 48 h	0 – 72 h
Control		0.0578	0.0694	0.0712
		0.0578	0.0728	0.0738
		0.0671	0.0687	0.0706
		0.0578	0.0679	0.0674
		0.0578	0.0687	0.0701
		0.0578	0.0741	0.0738
	Mean value	0.0593	0.0703	0.0711
	SD	0.0038	0.0026	0.0024
100 mg/L(nominal)		0.0578	0.0644	0.0631
		0.0578	0.0722	0.0727
		0.0578	0.0694	0.0731
		0.0578	0.0709	0.0740
		0.0578	0.0715	0.0708
		0.0578	0.0694	0.0699
	Mean value	0.0578	0.0696	0.0706
	SD	0.0000	0.0028	0.0040

Table 4: Growth Rates (r) and percentage inhibition of r during the test period

Test Group	Growth Rates (r)and % Inhibition ofr
	0–24 h		0–48 h		0–72 h
	r	%	r	%	r	%
Control	0.0593	-	0.0703	-	0.0711	-
100 mg/L(nominal)	0.0578	2.6	0.0696	0.9	0.0706	0.8

Table 5: Yield (y) and percentage inhibition of y during the test period

Test Group	Yield (y) and % Inhibition of y
	0–72 h
	y	%
Control	168.8	-
100 mg/L(nominal)	165.2	2.2

Table 6: The section-by-section specific growth rates in the control cultures

Section-by-Section Growth Rate
Expressed in hours	0–24 h	24-48 h	48-72 h
	0.0593	0.0812	0.0729
Expressed in days	0-1 day	1-2 day	2-3 day
	1.4235	1.9488	1.7500

Table 7: Concentration of Test Item measured in the Test Solutions

Nominal concentration (mg/L)	Start (June 05, 2018)		End (June 08, 2018)
	Concentration (mg/L)	% of the nominal concentration	Concentration (mg/L)	% of the nominal concentration
Control	not detected	-	not detected	-
100	97.5	97	0*	-
	99.1	99	26.3	26
	97	97	15.3	15
	98.5	98	20.3	20
	97.8	98	0*	-
	95.9	96	1.0*	-
Mean:	97.6	98	13	-
RSD:	1	1	-	-

* Below the LOQ.

LOQ = 10 mg/L, because sample was tenfold diluted.

For the calculation of the mean value LOQ/2 (5 mg/L) was entered.

Validity criteria fulfilled:

yes

Conclusions:

In a Growth Inhibition Test according to OECD TG 201 the 72-h EC10 and EC50 (growth rate and yield) of the test item was determined to be > 100 mg/L (nominal).

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to the principles of GLP, OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. A limit test was performed in which exponentially growing algal cells were exposed for 72 hours to aqueous test media (OECD Medium) containing the test item at the limit test concentration of 100 mg/L (6 replicates). In addition, a negative control and a positive control (with Potassium dichromate) with each 6 replicates was performed. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The algal cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. The initial cell density was about 104cells/mL in each test flask. Concentrations of the test item were determined using a LC/MS/MS method. Geometric mean exposure concentration was determined as 35.62 mg/L. Thus, the analytically measured test item concentration was below 80 % of the nominal at the end of the test (13 %). However, due to the test item characteristic keeping the concentration within ± 20 % of the nominal was not possible (semi-static or flow through system is not applicable in alga test). Therefore, biological endpoints were based on the nominal test item concentration. The results showed that the 0-72 h average specific growth rate and yield at the treatment group (100 mg/L nominal concentration) was not significantly different from the untreated control (directly from the raw data). Statistical comparisons of average specific growth rates and yield in control and in treated group were not necessary. The EC10, EC20, EC50, NOEC and LOEC values were given directly from the raw data. Based on nominal test item concentration the 72 -h EC10, EC10, EC50 and the LOEC values were determined to be > 100 mg/L and >35.62 mg/L based on geom. mean measured concentration. The 72-h NOEC was determined to be 100 mg/L (nominal) and 35.62 mg/L (geom. mean measured), respectively. All validity criteria of the guidelines were met and therefore the study is considered as valid.

Description of key information

In a Growth Inhibition Test according to OECD TG 201 the 72-h EC10 and EC50 (growth rate and yield) of the test item was determined to be > 100 mg/L (nominal).

Additional information

Key study

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to the principles of GLP, OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. A limit test was performed in which exponentially growing algal cells were exposed for 72 hours to aqueous test media (OECD Medium) containing the test item at the limit test concentration of 100 mg/L (6 replicates). In addition, a negative control and a positive control (with Potassium dichromate) with each 6 replicates was performed. The algal cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. Concentrations of the test item were determined using a LC/MS/MS method. In result, the EC10, EC20, EC50, NOEC and LOEC values were given directly from the raw data. Based on nominal test item concentration the 72 -h EC10, EC10, EC50 and the LOEC values were determined to be > 100 mg/L and >35.62 mg/L based on geom. mean measured concentration. The 72-h NOEC was determined to be 100 mg/L (nominal) and 35.62 mg/L (geom. mean measured), respectively. All validity criteria of the guidelines were met and therefore the study is considered as valid (Toxi-Coop ZRT, 2018).

Supporting study

The short-term toxicity of the test item to green algae was calculated using ECOSAR v1.11 class-specific estimation for Aldehydes (Mono). The test item falls in the applicability domain of the model. In result, the 96-h LC50 was calculated to be 7572.364 mg/L.

Disclaimer: In accordance with Article 13 and taking into account the conditions set out in Annex XI of Regulation (EC) No 1907/2006, information on intrinsic properties of substances might be generated by alternative methods other than tests if the (Q)SAR model has been proven to be valid, the substance falls in the applicability domain of the model, results are sufficient for the classification and labeling and/or risk assessment and the applied method is reliably and adequately documented. These criteria are considered to be fulfilled and, hence, the result to be suitable for classification and labelling purposes and/or risk assessment (SCC GmbH, 2018).

Conclusion

Based on results of a GLP-compliant guideline study (Toxi-Coop ZRT, 2018) the 72-h EC10 and EC50 (growth rate and yield) is considered to be > 100 mg/L (nominal). This result is supported by an ECOSAR estimation (SCC GmbH, 2018) since the calculated 96-h EC50 of the test item was even higher (96-h LC50 = 7572.364 mg/L, ChV = 1142.977 mg/L).