3-aminophenol

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:

From August 26, 2013 to February 26, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed according to the standard guideline.

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:

Short term toxicity test was performed according to the OECD guideline 201 and EU method C.3.

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations/Sampling method: Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period).
- Sample storage conditions before analysis: All samples were diluted by a factor of two with acetonitrile directly after sampling. All samples were centrifuged before analysis, in order to remove the algae. All samples were stored in a freezer (≤ - 10 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 51.2 mg test item into 512 mL
test water by intense stirring for 10 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations. The test media were prepared just before introduction of the algae (=start of the test).
- Eluate: no
- Differential loading: no
- Controls: In the control, test water was used without addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The appearance of the test item in the test media was determined daily in all test item concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): The test was started (0 hours) by inoculation of a biomass of 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in the laboratories of IBACON under test conditions.

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.

Test temperature:

21-22°C

pH:

pH was 8.0 at test start and 7.8 to 9.4 at test end

Nominal and measured concentrations:

Nominal: 100, 32, 10, 3.2, 1.0, 0.32 and 0.1 mg test item/L and a control.
At the start of the test 104% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 100% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 102% of nominal. Therefore, all reported results refer to nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel, Material, size, headspace, fill volume: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium.
- Type (delete if not applicable): The flasks were covered with glass dishes and incubated in a water bath.
- Aeration: Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks.
- Type of flow-through (e.g. peristaltic or proportional diluter): n.a.
- Renewal rate of test solution (frequency/flow rate): n.a.
- Initial cells density: 5000 algal cells per mL
- Control end cells density: 106 0000 cells per mL (212-fold increase)
- No. of organisms per vessel: 5000*50 algal cells per vessel
- No. of vessels per concentration (replicates): three replicates per test concentration
- No. of vessels per control (replicates): six replicates in the control
- No. of vessels per vehicle control (replicates): n.a.
Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometrical measurements. The additional replicates were incubated under the same conditions.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD Medium
Analytical grade salts were added at the following nominal concentrations in deionised water (conductivity <5 μScm-1):
Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace Elements:
Na2EDTA × 2 H2O 100.0 μg/L
FeCl3 × 6 H2O 64.0 μg/L
MnCl2 × 4 H2O 415.0 μg/L
H3BO3 185.0 μg/L
Na2MoO4 × 2 H2O 7.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 × 6 H2O 1.5 μg/L
CuCl2 × 2 H2O 0.01 μg/L

- Total organic carbon: /
- Particulate matter: /
- Metals: /
- Pesticides: /
- Chlorine: /
- Alkalinity: /
- Ca/mg ratio: Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
- Conductivity:/
- Culture medium different from test medium: no
- Intervals of water quality measurement: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The pH was measured in all test item concentrations and the control at the start and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The pH was adjusted in the test water with 2 M HCl to pH 8.0. No further adjustment.
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 6587 lux (range: 5940 to 7130 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: by spectrophotometrical measurement. Therefore defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). The cell densities in a number of samples from one control replicate were counted by microscope after 72 hours of test duration. Out of this control six defined dilutions were prepared and measured spectrophotometrically. Based on the counted cell densities and the absorption of the control sample and the dilutions a linear regression was performed for the calculation of the cell densities in all other samples measured spectrophotometrically during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125 and 3.2
- Justification for using less concentrations than requested by guideline: n.a.
- Range finding study: yes
- Test concentrations: The solubility pre-experiments and the range-finding test were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate, at least twice a year

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

67.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 56.1-76.6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

37.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 24.5-47.7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

46.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 33.4-56.5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Remarks on result:

other: Other details not known

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 35.8-125

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

24.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 6.17-28.4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

29.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 17.7-32.8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.387 mg/L [1.214 - 1.616], growth rate

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by logit analysis. For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Bonferroni-Welch t-test (yield and growth rate). The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

Cell Density: Increase in Control Cultures: 212-fold increase within 72h; CV of Sectional (Daily) Growth Rates in Control Cultures: 33.1 %; CV of Average Growth between Control Replicates: 1.3 %

Conclusions:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour EyC50 was calculated to be 41.0 mg test item/L and the 72-hour ErC50 value was calculated to be 67.7 mg test item/L. The 72-hour NOEyC was determined to be 10.0 mg test item/L and the associated 72-hour LOEyC was 32 mg test item/L. The 72-hour NOErC was determined to be 10.0 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L.

Executive summary:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test, according to OECD 201 guideline and EC method C.3, and GLP. Exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. This study encompassed 8 treatment groups (7 dose rates of the test item 100, 32, 10, 3.2, 1.0, 0.32 and 0.1 mg test item/L and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement. At the start of the test 104% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 100% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 102% of nominal. Therefore, all reported results refer to nominal concentrations. The 72-hour EyC50 was calculated to be 41.0 mg test item/L and the 72-hour ErC50 value was calculated to be 67.7 mg test item/L. The 72-hour NOEyC was determined to be 10.0 mg test item/L and the associated 72-hour LOEyC was 32 mg test item/L. The 72-hour NOErC was determined to be 10.0 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L. All the validity criteria were fulfilled.

Description of key information

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test, according to OECD 201 guideline and EC method C.3, and GLP. Exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. This study encompassed 8 treatment groups (7 dose rates of the test item 100, 32, 10, 3.2, 1.0, 0.32 and 0.1 mg test item/L and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement. At the start of the test 104% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 100% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 102% of nominal. Therefore, all reported results refer to nominal concentrations. The 72-hour EyC50 was calculated to be 41.0 mg test item/L and the 72-hour ErC50 value was calculated to be 67.7 mg test item/L. The 72-hour NOEyC was determined to be 10.0 mg test item/L and the associated 72-hour LOEyC was 32 mg test item/L. The 72-hour NOErC was determined to be 10.0 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L. All the validity criteria were fulfilled.

Key value for chemical safety assessment

EC50 for freshwater algae:

67.7 mg/L

Additional information

Based on the experimental data from various database and peer reviewed journals for the target chemical study have been reviewed to determine the mode of action of test chemical on the growth rate, biomass and behavioral changes of aquatic algae and cyanobacteria. The studies are as mentioned below:

 

In the first key study from experimental report 2015. The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test, according to OECD 201 guideline and EC method C.3, and GLP. Exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. This study encompassed 8 treatment groups (7 dose rates of the test item 100, 32, 10, 3.2, 1.0, 0.32 and 0.1 mg test item/L and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrically measurement. At the start of the test 104% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 100% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 102% of nominal. Therefore, all reported results refer to nominal concentrations. The 72-hour EyC50 was calculated to be 41.0 mg test item/L and the 72-hour ErC50 value was calculated to be 67.7 mg test item/L. The 72-hour NOEyC was determined to be 10.0 mg test item/L and the associated 72-hour LOEyC was 32 mg test item/L. The 72-hour NOErC was determined to be 10.0 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L. All the validity criteria were fulfilled.

 

First key study was supported by the second study from authoritative database 2018. Short term toxicity to green algae study was carried out for 72 hrs. The study was based on the effects of the test compound on green algae in a static fresh water system. Based on effect on growth rate of the test organism green algae, the 72 hr EC50 and NOEC value was determined to be 160 and 25 mg/l, and on the basis of biomass and area under the growth curve, the 72 hr EC50 and NOEC value was determined to be 62 and 25 mg/l, respectively. Thus, based on the EC50 value (62 mg/l), it can be concluded that the substance 3 -aminophenol can be considered as toxic to aquatic organisms and thus can be considered to be classified as aquatic chronic 3 as per the CLP classification criteria.

 

Based on the overall studies for the toxicity of test chemical on the growth of aquatic algae, chemical was consider as toxic and classified as aquatic chronic 3 as per the CLP classification criteria.