1-amino-4-hydroxy-2-phenoxyanthraquinone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Only few details on the study are available. Test conditions unavailable. Result considered plausible for a risk assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

not specified

Water media type:

not specified

Total exposure duration:

72 h

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 19 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

ca. 115 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 18 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 330 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 330 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Validity criteria fulfilled:

not specified

Conclusions:

EC50 (72 h) based on growth rate = 330 mg/l and EC50 (72 h) based on biomass = 19 mg/l. Due to the low purity of test material (ca. 20 %)

Executive summary:

The toxicity of the test substance to algae was determined by a 72 h test on Scenedesmus subspicatus according to OECD Test guideline 201, Growth Inhibition Test. Only few details of this test are available.

Biomass was observed as fluorescence, the curve was integrated and growth rate at the end of test concentrations refer to the test substance.

The effect levels, based on biomass, were the following:

EC10 = 3.1 mg/l, EC50 = 19 mg/l and EC90 = 115 mg/l;

while effect levels, based on growth rate, were as follows:

EC10 = 18 mg/l, EC50 = 330 mg/l and EC90 > 330 mg/l.

Concentrations refer to active ingredient.

Based on effects on growth rate, the substance is considered as non toxic to aquatic algae.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

from April 16 to May 20, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The complete read across justification is detailed in section 13. Source study has reliability 1.

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100 % v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-finding test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.0021 mg/l could be obtained using a saturated solution method of preparation.

The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution for a period of 72 hours.

A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10 % v/v saturated solution. An aliquot (450 ml) of each of the stock solutions was separately inoculated with algal suspension (4.9 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution.

Definitive test
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution.

An aliquot (1 liter) of the stock solution was inoculated with 6.7 ml of algal suspension to give the required test concentration of 100 % v/v saturated solution.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection):Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.
- Method for cultivation : Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1E+04 - 1E+05 cells/ml.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

Temperature within the incubator was recorded daily.

pH:

pH of the control and 100 % v/v saturated solution test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter.

Nominal and measured concentrations:

Range-finding test: nominal test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution.
Definitive test: nominal test concentration of 100 % v/v saturated solution.

Details on test conditions:

Preliminary Media Preparation Trial
Information provided by the Sponsor indicated the water solubility of the test item to be 0.254 µg/l. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultra sonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.


Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.0021 mg/l could be obtained using a saturated solution method of preparation.

The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution for a period of 72 hours.

A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10 % v/v saturated solution. An aliquot (450 ml) of each of the stock solutions was separately inoculated with algal suspension (4.9 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All 0-Hour samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100 % v/v saturated solution to confirm that at the highest attainable test concentration no effect on algal growth was observed.


Experimental Preparation
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution.

An aliquot (1 liter) of the stock solution was inoculated with 6.7 ml of algal suspension to give the required test concentration of 100 % v/v saturated solution.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Exposure Conditions
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of solution were used for the control and 100 % v/v saturated solution treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.44 x 1E+05 cells per ml. Inoculation of 1 liter of test medium with 6.7 ml of this algal suspension gave an initial nominal cell density of 5 x 103 cells per ml and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Evaluations
Test Organism Observations
Samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (1n Nn – 1n N1) / (tn – t1)

Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = ((µc - µt) / µc) x 100

Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = ((Yc – Yt) / Yc) x 100

Where:

Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

Determination of ECx Values
ECx values were determined by inspection of the growth rate and yield data after 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Remarks on result:

other: no toxic effects at saturation

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution. 
 
Based on this information a single test concentration of six replicates, of 100 % v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.
 
Chemical analysis of the 100 % v/v saturated solution test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0036 mg/l. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.
 
 
Definitive Test
Growth Data From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h): >100 % v/v saturated solution
ErC20 (0 - 72 h): >100 % v/v saturated solution
ErC50 (0 - 72 h): >100 % v/v saturated solution

where ErCx is the test concentration that reduced growth rate by x %.

Statistical analysis of the growth rate data was carried out for the control and 100 % v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P≥0.05), between the control and 100 % v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 % v/v saturated solution.

Inhibition of Yield
EyC10 (0 - 72 h: > 100 % v/v saturated solution
EyC20 (0 - 72 h): > 100 % v/v saturated solution
EyC50 (0 - 72 h): > 100 % v/v saturated solution

Where:

EyCx is the test concentration that reduced yield by x %.

Statistical analysis of the yield data was carried out. There were no statistically significant differences (P≥0.05), between the control and 100 % v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 % v/v saturated solution.

Results with reference substance (positive control):

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.2 mg/l; 95 % confidence limits 1.1 – 1.4 mg/l
EyC50 (0 – 72 h): 0.63 mg/l; 95 % confidence limits 0.57 – 0.70 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 % v/v saturated solution test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata were investigated and gave EC50 values greater than 100 % v/v. The No Observed Effect Concentration was 100 % v/v saturated solution.
This study showed that there were no toxic effects at saturation.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was described in the OECD guideline 201 (2006) referenced as EU method C.3 (2009).

 

A preliminary range-finding test showed no effects on growth rate at concentrations 0.10, 1.0, 10 and 100 % v/v saturated solution.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Based on this information, a single concentration of 100 % v/v saturated solution was selected for the definitive test.

The test item solution was prepared by stirring an excess (50 mg/l) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test item.

This experimental design conforms to a limit test, to assess effect on growth rate at the highest attainable concentration.

In the definitive test, performed in six replicates, algae were exposed to test item for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. 

   

Under test conditions, EC50 value greater than 100 % v/v saturated solution and No Observed Effect Concentration of 100 % v/v saturated solution were found.

This study showed that there were no toxic effects at saturation.

Description of key information

EC50 > 100 % v/v saturated solution, NOEC = 100 % v/v saturated solution.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Due to the lack of details in the available study on Disperse Red 060, a read across approach was used in order to complete the assessment for toxicity to aquatic algae. A detailed description of the read across chosen is reported in section 13.

The toxicity of Disperse Red 060 to algae and cyanobacteria was determined according to OECD guideline 201, Growth Inhibition Test. Two studies were available.

In the first study, aquatic algae, i.e. Scenedesmus subspicatus, were exposed to test substance for 72 hours. Only a short abstract was available. Observed parameters were biomass as fluorescence, curve integrated and growth rate at the end of the test. Reported concentrations refer to test substance. The effect levels, based on biomass, were the following: EC10 = 3.1 mg/l, EC50 = 19 mg/l and EC90 = 115 mg/l; while effect levels, based on growth rate, were as follows: EC10 = 18 mg/l, EC50 = 330 mg/l and EC90 > 330 mg/l.

In the second study, aquatic algae, i.e. Pseudokirchneriella subcapitata, were exposed to Similar Substance 01 for 72 hours using a nominal concentration of 100 % v/v saturated solution. In principle, this setup may be associated to a limit test. No toxic effects were seen at the highest attainable concentration.