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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From December 24 2010 to January 04, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details for read across approach are included into the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the concentrations of the test item Blue were determined in the duplicate test medium samples from the nominal test concentrations of 10 to 100 mg/l.
- Number of samples: duplicate samples were taken from the test media of all test concentrations and control.
- Sampling time: at the start of the test (without algae) and at the end of the test (containing algae).
- Sample storage conditions before analysis: all samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis.

Vehicle:

no

Details on test solutions:

The test medium of the highest nominal concentration of 100 mg/l was prepared by dissolving 100.71 mg of the test item completely in 1000 ml of test water using intense stirring for 10 minutes at room temperature. The test medium of the highest test concentration was serially diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).

BREEDING
- Conditions: the algae were cultivated at testing laboratories under standardized conditions according to the test guidelines.
- Inoculumn: an inoculum culture was set up three days before the start of the exposure.
- Growth phase: the algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The water temperature during the test was maintained at 22 °C.

pH:

At the start of the test, the pH measured in the treatments was between 8.0 and 8.1. At the end of the test, pH values of 7.9 to 8.1 were measured.

Nominal and measured concentrations:

1.0, 3.2, 10, 32 and 100 mg/l (nominal)
The highest concentration of 100 mg/l corresponded to 72 mg dry matter/l due to a 28 % water content of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: since the test item caused a dark colored solution in the test water, 17 ml of test solution were placed in 100-ml Erlenmeyer flasks achieving a reduced layer thickness of 0.9 cm and enhancing the light supply for the algae.
- Type: each test flask was covered with a glass dish.
- Stirring: during exposure, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density: 10000 cells/ml.
- No. of vessels per concentration: three replicates.
- No. of vessels per control: six replicates.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.
- Macro-nutrients: NaHCO3 50.0 mg/l; KH2PO4 1.6 mg/l; MgSO4 × 7 H2O 15.0 mg/l; MgCl2 × 6 H2O 12.0 mg/l; CaCl2 × 2 H2O 18.0 mg/l; NH4Cl 15.0 mg/l.
- Trace elements: H3BO3 185.0 μg/l; MnCl2 × 4 H2O 415.0 μg/l; ZnCl2 3.0 μg/l; CoCl2 × 6 H2O 1.5 μg/l; CuCl2 × 2 H2O 0.01 μg/l; Na2MoO4 × 2 H2O 7.0 μg/l; FeCl3 × 6 H2O 64.0 μg/; Na2EDTA × 2 H2O 100.0 μg/l.
- Hardness: 0.24 mmol/l (= 24 mg/l as CaCO3).

OTHER TEST CONDITIONS
- Illumination: fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks.
- Light intensity and quality: the mean measured light intensity at the level of the test solutions was approximately 7400 Lux (range: 6820 to 7900 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within a ± 15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED
The algal biomass in the samples was determined by fluorescence measurement. The measurements were performed at least in duplicate.
At the end of the test, a sample was taken from the control and from the test concentration of nominal 32 mg/l to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected. This test concentration was chosen since the algal cell density was too low for a reliable examination at the highest nominal concentration of 100 mg/l.

MEASUREMENTS
- Shading effect: the shading effect for each treatment was determined by measurement of the light intensity under each test solution.
- pH: measured and recorded in each treatment at the start and end of the test.
- Temperature: measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
- Medium appearance: appearance of the test media was also recorded daily.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: the selection of the test concentrations was based on the results of range-finding tests (non-GLP).
- Storage stability: in pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the concentration of 32 mg/l and at all higher test concentrations (results of Williams t-tests, one-sided, α = 0.05). Thus, this concentration was determined to be the 72-hour LOEC.
The test design was adjusted to the characteristics of the test item (dark coloured solution). However, it cannot be excluded, that the adverse effect of the test item on the growth of Pseudokirchneriella subcapitata at the two highest test concentrations of 32 and 100 mg/l was caused to a great extent by the significantly decreased light intensity (27.5 and 3.3 % of the control value, respectively) at these test concentrations.
The 72-hour NOEC was determined to be 10 mg/l, since up to and including this test concentration, the growth rate and yield of the algae after 72 hours were not statistically significantly lower than in the control.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 32 mg/l and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

CONTROL
In the control, the biomass increased by a factor of 170 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 10.5 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 2.5 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7 %. Thus, the validity criterion was fulfilled.

APPEARANCE OF TEST MEDIUM
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions coloured by the test item throughout the test period.

MEASURED CONCENTRATIONS
The measured concentrations of test item in the test media of the test concentrations of 10 to 100 mg/l were between 91 and 97 % of the nominal values at the start of the test and between 83 and 85 % at the end of the test. Thus, the correct dosing of the test item was confirmed. The test item was sufficiently stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations (including water content) of the test item.

Results with reference substance (positive control):

The result of the latest positive control test performed in October 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.99 mg/l, range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71-1.7 mg/l).

Average Growth Rates (μ)

Nominal test item concentration (mg/l)	Average growth rate μ (day-1) and inhibition of μ (Ir)
	0 - 24 hrs		0 - 48 hrs		0 - 72 hrs
	μ	Ir(%)	μ	Ir(%)	μ	Ir(%)
Control	1.81	0.0	1.74	0.0	1.71	0.0
1.0	1.91	-5.5	1.77	-1.6	1.70	0.9
3.2	1.96	-7.8	1.78	-2.1	1.72	-0.7
10	1.75	3.4	1.65	5.0	1.74	-1.9
32	1.14	37.2	1.20	30.8	1.41*	17.7
100	0.03	98.4	0.38	78.2	0.81*	52.5

*: mean value significantly lower than in the control (according to Williams t-test, one-sided, α = 0.05)

Section-by-Section Growth Rates

Nominal test item concentration (mg/l)	Section-by-section growth rates (day-1) and inhibition of the growth rates (Ir)
	0 - 24 hrs		24 - 48 hrs		48 - 72 hrs
	μ	Ir(%)	μ	Ir(%)	μ	Ir(%)
Control	1.81	0.0	1.67	0.0	1.65	0.0
1.0	1.91	-5.5	1.62	2.6	1.55	6.1
3.2	1.96	-7.8	1.60	4.1	1.61	2.4
10	1.75	3.4	1.56	6.7	1.92	-16.4
32	1.14	37.2	1.27	23.8	1.81	-10.0
100	0.03	98.4	0.73	56.3	1.68	-1.9

Yield (Y)

Nominal test item concentration (mg/l)	Yield Y (x 10^3) and inhibition of Y (Iy)
	0 - 24 hrs		0 - 48 hrs		0 - 72 hrs
	Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	7	0.0	43.3	0.0	230.8	0.0
1.0	7.9	-12.3	45.9	-6.0	219.8	4.7
3.2	8.3	-17.9	46.4	-7.1	239.7	-3.9
10	6.5	7.3	36.0	16.9	253.2	-9.7
32	2.9	58.6	13.9	68.0	92.5 *	59.9
100	0.0	99.4	1.5	96.4	14.2 *	93.8

*: mean value significantly lower than in the control (according to Williams t-test, one-sided, α = 0.05)

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (72h): 93 mg/l (nominal)
EyC50 (72h): 28 mg/l (nominal)

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test, according to the OECD guideline 201 (2006) and the Commission Regulation (EC) No. 761/2009, C.3. Test item concentrations of 1.0, 3.2, 10, 32 and 100 mg/l (nominal) were tested alongside a control. The highest concentration of 100 mg/l corresponded to 72 mg/l dry matter due to a 28 % water content of the test item. Since the test item caused a dark coloured solution in the test water, 17 ml of test solution were placed into 100 ml Erlenmeyer flasks, achieving a reduced layer thickness of 0.9 cm and enhancing the light supply for the algae.

The measured test item concentrations in test media of 10 to 100 mg/l were between 91 % and 97 % of the nominal values at the start of the test, and between 83 % and 85 % at the end of the test. Therefore, the correct dosing of the test item was confirmed. The test item was sufficiently stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations (including water content) of the test item. The test design was adjusted to the characteristics of the test item (dark coloured solution). However, adverse effects of the test item on the growth of Pseudokirchneriella subcapitata at the two highest test concentrations of 32 and 100 mg/l was greatly influenced by the significantly decreased light intensity (27.5 % and 3.3 % of the control value, respectively) at these test concentrations.

The E_rC₅₀ was identify to be 93 mg/l, while the E_yC₅₀ was recorded at 28 mg/l. The No Observed Effect Concentration (NOEC) was identified as 10 mg/l, on the basis of both growth rate and yield.

Conclusion

E_rC₅₀ (72h): 93 mg/l (nominal)

E_yC₅₀ (72h): 28 mg/l (nominal)

NOEC (72h): 10 mg/l (nominal)

Description of key information

NOEC (72h, Pseudokirchneriella subcapitata, growth and yield) = 10 mg/l (nominal)

LOEC (72h, Pseudokirchneriella subcapitata, growth inhibition) = 32 mg/l (nominal)

E_rC₅₀ (72h): 93 mg/l (nominal)

E_yC₅₀ (72h): 28 mg/l (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

93 mg/L

Additional information

No studies on the toxicity to algae and cyanobacteria are available for Direct Blue 267. Nevertheless, a study conducted on the structural analogue Similar Substance 01 is available; the read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

The influence of the Similar Substance 01 on the growth of the freshwater green algae was investigated in a 72-hour static test according to the OECD guideline 201 (2006). The test item had a statistically significant inhibitory effect on the growth of the algae after 72 hours of exposure at 32 mg/l and above, therefore, this concentration was determined to be the 72-hour LOEC. The test design was adjusted to the characteristics of the test item (dark coloured solution). However, it cannot be excluded that the adverse effect of the test item on the growth of Pseudokirchneriella subcapitata at the two highest test concentrations of 32 and 100 mg/l was caused to a great extent by the significantly decreased light intensity (27.5 % and 3.3 % of the control value, respectively) at these test concentrations. The 72-hour NOEC was determined to be 10 mg/l, since up to and including this test concentration, the growth rate and yield of the algae after 72 hours were not statistically significantly lower than in the control. The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 32 mg/l and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions coloured by the test item throughout the test period.

The measured concentrations of test item in the test media of the test concentrations of 10 to 100 mg/l were between 91 % and 97 % of the nominal values at the start of the test and between 83 % and 85 % at the end of the test, showing that the test item was sufficiently stable in the test media over the test period of 72 hours (Kimmel, 2011).