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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:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 12 March 2010; Experiment completion date - 23 March 2010; Study completion date - 27 May 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9% all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Analytical monitoring:

yes

Details on sampling:

For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control.

For the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 mL) was too small to perform the analyses.

All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In a pre-test (non-GLP) the samples proved to be stable under these storage conditions.

Vehicle:

no

Details on test solutions:

The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 103.76 mg of the test item completely in 1038 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was 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):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) Nygaard et al (1986) recommended describing the taxa within the Genus Raphidocelis HINDAK as: Raphidocelis subcapitata (KORSIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSIKOV
Syn.: Kirchneriella subcapitata KORSIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1
∙ NYGAARD, G., KOMÁREK, J., KRISTIANSEN, J., SKULBERG O.M. (1986). Taxonomic designations of the bioassay alga NIVA-CHL 1 ("Selenastrum capricornutum") and some related strains. Opera Botanica 90:5-46
- Strain: Strain No. 61.81 SAG
- Source: supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.
- Culturing media and conditions: Same as test
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C.

pH:

Start: 7.9 - 8.0, End: 7.9 - 8.2

Dissolved oxygen:

During exposure, the test solutions were continuously stirred by magnetic stirrers.

Salinity:

Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing (see Table M1 below)

Nominal and measured concentrations:

0, 1.0, 3.2, 10, 32 and 100 mg/L nominal

The concentrations of the test item test substance were determined in the duplicate test medium samples from the nominal test concentrations of 3.2 to 100 mg/L. The samples from the nominal test concentration 1.0 mg/L were not analyzed, since this concentrations was below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.

The measured concentrations of the test item in the test media of the test concentrations of 3.2 to 100 mg/L were between 86 and 106% of the nominal values at the start and the end of the test. Thus, the correct dosing of the test item test substance at test start was confirmed. Furthermore, it has been demonstrated that the test item was stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks
- Type: Each test flask was covered with a glass dish.
- Material, size, headspace, fill volume: Since the test water was significantly colored by the test item, 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.
Aeration: During exposure, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density: 10'000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile purified water


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Temperature: The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C
- Photoperiod: 24 h / day
- Light intensity and quality: The test flasks were illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7960 Lux (range: 6970 to 8650 Lux, measured at nine places in the experimental area). The light intensity was within ± 15% from the average light intensity as recommended by the guideline. The shadowing effect of each test concentration was determined by measurement of the light intensity under each test solution: In the nominal test item concentration (mg/L) of 0, 1.0, 3.2, 10, 32, and 100 mg/L the Light intensity (Lux) was 8300, 8360, 8230, 7950, 7710, and 6990 respectively.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: The algal biomass in the samples was determined by measurement of the algal cell density using an electronic particle counter (Coulter Counter, Model ZM). 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 100 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.
- The pH was measured and recorded in each treatment at the start and at the end of the test.
- The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
- The appearance of the test media was also recorded daily.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: Based on the range-finding study

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 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
- 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 100 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.
- In the control the biomass increased by a factor of 128 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 16%. 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 1.0%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.
- No remarkable observations were made concerning the appearance of the test media. All test media were clear orange colored solutions throughout the test period.
- At the start of the test, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values of 7.9 to 8.2 were measured. The water temperature during the test was maintained at 22 °C.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- EC50: 0.75 mg/L (72h) (Sept 2009)
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in September 2009 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.75 mg/L (study C63678), range of the 72-hour EC50 for the growth rate from 2000 to 2009: 0.71 - 1.7 mg/L).

Reported statistics and error estimates:

The R² fit of the calibration curve used was 1.0000.

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 two highest test concentrations of 32 mg/L and 100 mg/L (results of Williams tests, one-sided, α = 0.05, Table 2 and Table 3). Thus, this concentration was determined to be the 72-hour LOEC.

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 tabulated values represent rounded results obtained by calculation using the exact raw data.

 

Table 1: Biomass of Algae

Nominal test item concentration [mg/L]	Rep. no.	Biomass of algae*
		24 hours	48 hours	72 hours
Control	1	9.0	44.0	298.9
	2	10.4	53.5	321.9
	3	10.5	64.6	299.0
	4	8.3	44.6	306.4
	5	9.5	58.7	287.3
	6	8.3	52.2	329.0
	Mean	9.3	52.9	307.1
	SD	1.0	8.0	15.6
1.0	1	9.2	53.1	245.7
	2	9.5	43.8	341.1
	3	8.2	53.4	321.4
	Mean	8.9	50.1	302.7
	SD	0.7	5.5	50.4
3.2	1	9.4	50.6	236.9
	2	9.9	53.2	299.6
	3	8.3	59.6	289.8
	Mean	9.2	54.5	275.4
	SD	0.8	4.6	33.7
10	1	9.8	44.3	238.6
	2	7.4	41.4	354.8
	3	7.8	48.4	287.7
	Mean	8.4	44.7	293.7
	SD	1.3	3.5	58.3
32	1	6.0	35.3	226.4
	2	5.4	33.3	312.8
	3	6.3	33.8	233.3
	Mean	5.9	34.1	257.5
	SD	0.5	1.1	48.0
100	1	2.8	16.9	225.7
	2	2.5	16.3	234.7
	3	2.2	16.6	227.0
	Mean	2.5	16.6	229.1
	SD	0.3	0.3	4.9

SD: Standard deviation

* The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units ( ∙ 10³). At the start of the test, the initial cell density was 10’000 algal cells/mL, corresponding to 2.40 x 10³ relative fluorescence units.

 

Table 2: Average Growth Rates (µ)

Nominal test item concentration [mg/L]	Average growth rate µ [day^-1] and inhibition of µ (Ir)
	0-24 h		0-48 h		0-72 h
	µ	Ir[%]	µ	Ir[%]	µ	Ir[%]
Control	1.35	0.0	1.54	0.0	1.62	0.0
1.0	1.31	3.0	1.52	1.6	1.61	0.5
3.2	1.34	1.0	1.56	-1.2	1.58	2.3
10	1.24	8.3	1.46*	5.2	1.60	1.2
32	0.90*	33.8	1.33*	14.0	1.55*	3.8
100	0.04*	97.3	0.97*	37.3	1.52*	6.0

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

---: not analyzed

 

 

Table 3: Yield(Y)

Nominal test item concentration [mg/L]	Yield Y ( ∙ 10³) and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	6.9	0.0	50.5	0.0	304.7	0.0
1.0	6.5	5.7	47.7	5.6	300.3	1.4
3.2	6.8	2.0	52.1	-3.0	273.0	10.4
10	6.0	14.0	42.3*	16.3	291.3	4.4
32	3.5*	49.6	31.7*	37.2	255.1*	16.3
100	0.1*	98.5	14.2*	71.9	226.7*	25.6

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

---: not analyzed

 

 

Table 4: 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 h		24-48 h		48-72 h
	µ	Ir [%]	µ	Ir [%]	µ	Ir [%]
Control	1.35	0.0	1.73	0.0	1.77	0.0
1.0	1.31	3.0	1.72	0.5	1.79	-1.5
3.2	1.34	1.0	1.78	-2.8	1.62	8.4
10	1.24	8.3	1.68	2.8	1.87	-5.9
32	0.90	33.8	1.76	-1.6	2.01	-13.8
100	0.04	97.3	1.90	-9.6	2.63	-48.6

---:         not analyzed

 

  

Table 5: pH Values in theTreatments

Nominal test item concentration [mg/L]	pH values
	Start	End
Control	8.0	7.9
1.0	8.0	8.0
3.2	8.0	8.2
10	7.9	8.2
32	7.9	8.2
100	7.9	8.1

Validity criteria fulfilled:

yes

Conclusions:

The test substance is considered not acute toxic to aquatic freshwater green algae.

Executive summary:

The influence of the test item test substance on the growth of a freshwater green algal species (Pseudokirchneriella subcapitata) was investigated in a 72-hour static test under GLP according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3.

The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control. The measured concentrations of the test item in the test media of the test concentrations of 3.2 to 100 mg/L were between 86 and 106% of the nominal values at the start and the end of the test. Thus, the correct dosing of the test item test substance at test start was confirmed. Furthermore, it has been demonstrated that the test item was stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

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 two highest test concentrations of 32 mg/L and 100 mg/L (results of Williams tests, one-sided, α = 0.05). Thus, this concentration was determined to be the 72-hour LOEC. 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. As not 50% effect level was reached, no EC₅₀ was established.

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 100 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 and including this concentration.

In the control the biomass increased by a factor of 128 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 16%. 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 1.0%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear orange coloured solutions throughout the test period. At the start of the test, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values of 7.9 to 8.2 were measured. The water temperature during the test was maintained at 22 °C.

In conclusion the test item test substance is considered not acute toxic to aquatic freshwater green algae.

Description of key information

The EC50 and the NOEC for growth rate were >100 and 10 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

The influence of the test item test substance on the growth of a freshwater green algal species (Pseudokirchneriella subcapitata) was investigated in a 72-hour static test under GLP according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3. The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control. The measured concentrations of the test item in the test media of the test concentrations of 3.2 to 100 mg/L were between 86 and 106 % of the nominal values at the start and the end of the test. Thus, the correct dosing of the test item test substance at test start was confirmed. Furthermore, it has been demonstrated that the test item was stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item. 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 two highest test concentrations of 32 mg/L and 100 mg/L (results of Williams tests, one-sided, α = 0.05). Thus, this concentration was determined to be the 72-hour LOEC. 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. As 50 % effect level was not reached, no EC₅₀ was established. 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 100 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 and including this concentration. In the control the biomass increased by a factor of 128 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 16 %. 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 1.0 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7 %. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear orange coloured solutions throughout the test period. At the start of the test, the pH measured in the treatments was between 7.9 and 8.0. At the end of the test, pH values of 7.9 to 8.2 were measured. The water temperature during the test was maintained at 22 °C. In conclusion the test item test substance is considered not acute toxic to aquatic freshwater green algae.