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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-Oct-2010 to 19-Nov-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

Materials and methods

Test guidelineopen allclose all
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
Principles of method if other than guideline:
Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared using ultrasonic treatment and intense stirring. After 24h, the dispersion was filtered. The dilutions 1:2, 1:6.3, 1:20, 1:63, 1:630 and 1:2000 were used as test media.
GLP compliance:
yes (incl. QA statement)
Remarks:
swissmedic: Date of inspection: 05 to 09-Nov-2007 and 26 to 30-Nov-2007; Date of decision: 2008-04-30, Date of signature: 12-Nov-2008

Test material

Constituent 1
Reference substance name:
Acid Yellow RN 2903
IUPAC Name:
Acid Yellow RN 2903
Constituent 2
Reference substance name:
Vers. Kilo 6, 26.05.2009
IUPAC Name:
Vers. Kilo 6, 26.05.2009
Details on test material:
The test item and the following information were provided by the Sponsor:

Identity: Acid Yellow RN 2903
Batch No.: Vers. Kilo 6, 26.05.2009
Purity: Approx. 65%
Expiration Date: 01-Aug-2015
Storage Conditions: At room temperature at about 20 °C, in the dark.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

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 x 15 mL) was too small to perform the analyses.

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

The concentrations of the test item Acid Yellow RN 2903 were determined in the duplicate test medium samples from all dilutions. From the control samples, one of the duplicate samples was analyzed per sampling time.

The analytical procedure and results are described in the attached Appendix I - Analytical Investigations.

Test solutions

Vehicle:
no
Details on test solutions:
Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 100.02 mg of the test item in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 5 minutes and intense stirring on a magnetic stirrer over 15 minutes, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. After the stirring period, the dispersion was left undisturbed for 24 hours in the dark to allow precipitation of the test item.

After the 24-hour resting period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm). The undiluted filtrate was used as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was further diluted with test water. The test media were prepared just before the start of the test (= start of exposure).

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

Nygaard et al. 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


An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).
Test temperature:
The water temperature during the test was 22 °C.
pH:
At the start of the test, the pH measured in the treatments was 8.1. At the end of the test, pH values of 8.4 to 9.5 were measured (see attached Table 6). The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test.
Dissolved oxygen:
not applicable
Salinity:
according to OECD test guideline
Nominal and measured concentrations:
The following treatments were tested: Dilution 1:2, 1:6.3, 1:20, 1:63, 1:200, 1:630 and 1:2000. Additionally, a control was tested in parallel (test water without test item).
At the start of the test, the analytically determined concentrations of Acid Yellow RN 2903 in the test media (dilutions 1:2.0, 1:6.3, 1:20, 1:63, 1:200, 1:630 and 1:2000) were 55, 17, 5.5, 1.8, 0.58, 0.19 and 0.050 mg/L, respectively (see analytical results and Table 2 in Appendix I). During the test period of 72 hours, a decrease of the test item concentration in the test media occurred. At the end of the test, the determined concentrations were 20, 10, 5.0, 1.7, 0.61, 0.17 and 0.040 mg/L.
For further information please see section " Any other information on materials and methods incl. tables" below.
Details on test conditions:
Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. For further details on the test water please see section " Any other information on materuals and methods incl. tables" below.

50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and 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 7500 Lux (range: 7060 to 8260 Lux, measured at nine places in the experimental area).

Since the test item caused a yellow colored solution in the test water, the shading effect for each treatment was determined by measurement of the light intensity under each test solution. For further information please see section " Any other information on materials and methods incl. tables" below.

The selection of the test concentrations was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item (non GLP).

The following treatments were tested: Dilution 1:2, 1:6.3, 1:20, 1:63, 1:200, 1:630 and 1:2000. Additionally, a control was tested in parallel (test water without test item).
The test design included three replicates per test concentration and six replicates of the control.
Reference substance (positive control):
yes
Remarks:
tested twice a year

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.59 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
56 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 49 - 65 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 3.3 – 4.8 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 2.1 – 2.9 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 0.24 – 0.54 mg/L
Duration:
72
Dose descriptor:
EC20
Effect conc.:
7.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 6.5 – 7.9 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 0.61 – 1.1 mg/L
Details on results:
At the start of the test, the analytically determined concentrations of Acid Yellow RN 2903 in the test media (dilutions 1:2.0, 1:6.3, 1:20, 1:63, 1:200, 1:630 and 1:2000) were 55, 17, 5.5, 1.8, 0.58, 0.19 and 0.050 mg/L, respectively. During the test period of 72 hours, a decrease of the test item concentration in the test media occurred. At the end of the test, the determined concentrations were 20, 10, 5.0, 1.7, 0.61, 0.17 and 0.040 mg/L.
The influence of the test item on the growth of the algae is shown in the attached Table 1 to Table 3 and in Figure 1 to Figure 3.
For further details on biological results please see section " Any other information on tesults incl. tables".

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 mean measured concentration of 0.59 mg/L and at all higher test concentrations (results of Dunnett t-tests, one-sided smaller, alpha = 0.05, see attached Table 2 and Table 3). Thus, this mean measured concentration was determined to be the 72 hour LOEC.
The 72 -hour NOEC was determined to be at the mean measured concentration of 0.18 mg/L. The growth rate and yield of the algae after 72 hours were lower than in the control at this concentration, but within the natural variance tolerated by the guidelines. The difference at this test item treatment to the control in growth rate and yield of the algae after 72 hours was below 10%, which is not seen as a biologically relevant effect.
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the dilution of 1:200 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 148 over 72 hours (see attached Table 1). 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, Table 4) during 72 hours was 17%. 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 0.6%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

The following observations were made concerning the appearance of the test media: The test medium of the dilution 1:2000 was a clear solution throughout the entire test duration, while the higher concentrated test media were yellow colored by the test item. The coloration of the 1:630 dilution dissipated after 24 hours. At all higher test concentrations the coloration was visible throughout the entire test period (see attached Table 5). However, the coloration had no influence on the light intensity during the test and therefore no shading effect .
Results with reference substance (positive control):
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 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 (study C97564), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71to 1.7 mg/L).

Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of, average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t test and Welch t test.

Any other information on results incl. tables

The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and end of the test:

 

Treatment / Dilution

Mean measured concentration of the test item
(geometric mean)

 

(mg/L)

(% of initially measured)

1:2000

0.045

90

1:630

0.18

95

1:200

0.59

103

1:63

1.8

99

1:20

5.3

96

1:6.3

13

77

1:2

33

60

The biological results can be summarized as follows (on the basis of mean measured concentrations of the test item):

 

Parameter

Growth rate

Yield

(0-72 h)

 

 

EC10  (mg/L)

2.5

0.38

95% confidence interval

2.1 – 2.9

0.24 – 0.54

EC20  (mg/L)

7.2

0.85

95% confidence interval

6.5 – 7.9

0.61 – 1.1

EC50  (mg/L)

56*

4.0

95% confidence interval

49 - 65

3.3 – 4.8

NOEC (mg/L)

0.18

0.18

LOEC (mg/L)

0.59

0.59

 

*:           extrapolated value

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
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 mean measured concentration of 0.59 mg/L and at all higher test concentrations (results of Dunnett t-tests, one-sided smaller, alpha = 0.05).
Executive summary:

The influence of the test item Acid Yellow RN 2903 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.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared using ultrasonic treatment and intense stirring. After 24h, the dispersion was filtered. The dilutions 1:2, 1:6.3,1:20, 1:63, 1:630 and 1:2000 were used as test media. Additionally, a control was tested in parallel.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

At the start of the test, the analytically determined concentrations of Acid Yellow RN 2903 in the test media (dilutions 1:2, 1:6.3,1:20, 1:63, 1:200, 1:630 and 1:2000) were 55, 17, 5.5, 1.8, 0.58, 0.19 and 0.050 mg/L, respectively. During the test period of 72 hours, a decrease of the test item concentration in the test media occurred. At the end of the test, the determined concentrations were 20, 10, 5.0, 1.7, 0.61, 0.17 and 0.040 mg/L. The biological results were related to the mean measured test item concentrations.

 

The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and end of the test:

 

Treatment

Mean measured concentration of the test item
(geometric mean)

(mg/L)

(% of initially measured)

Dilution 1:2000

0.045

90

Dilution 1:630

0.18

95

Dilution 1:200

0.59

103

Dilution 1:63

1.8

99

Dilution 1:20

5.3

96

Dilution 1:6.3

13

77

Dilution 1:2

33

60

 

The biological results can be summarized as follows (on the basis of mean measured concentrations of the test item):

 

Parameter

Growth rate

Yield

(0-72 h)

 

 

EC10  (mg/L)

2.5

0.38

95% confidence interval

2.1 – 2.9

0.24 – 0.54

EC20  (mg/L)

7.2

0.85

95% confidence interval

6.5 – 7.9

0.61 – 1.1

EC50  (mg/L)

56*

4.0

95% confidence interval

49 - 65

3.3 – 4.8

NOEC (mg/L)

0.18

0.18

LOEC (mg/L)

0.59

0.59

 *:           extrapolated value