3-aminophenol

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1996-01-31 to 1996-03-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed according to the guideline.

Qualifier:

according to guideline

Guideline:

other: OECD 202 Daphnia sp. acute immobilization test and reproduction test

Version / remarks:

1984

Deviations:

no

Principles of method if other than guideline:

Study was conducted to investigate the long term effect of test chemical on the growth of aquatic invertebrates according to OECD 202 Daphnia sp. acute immobilization test and reproduction test.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplier kept confidential; batch: GC01
- Expiration date of the lot/batch:not specified
- Purity test date: not specified

RADIOLABELLING INFORMATION (if applicable) na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Test substance was stored in a refrigerating chamber of this laboratory
- Stability under test conditions: the test substance was stable
- Solubility and stability of the test substance in the solvent/vehicle: na

TREATMENT OF TEST MATERIAL PRIOR TO TESTING no

Analytical monitoring:

yes

Details on sampling:

- Concentrations / Sampling method / Sample storage conditions before analysis :
For all test groups, HPLC analysis was carried out (1 sample/1 test group was prepared by mixing the same quantity of test solutions sampled from the middle layers of each of the 4 test vessels), at the beginning (day 0), day 3, day 7, day 14 and at the end of exposure (day 21), after removing food by centrifugation, as is or diluted.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test substance was added to dilution water, dissolved by ultrasonication procedure, and the solution of 1,000 mg/L was prepared. This solution was diluted with dilution water to prepare stock solutions of concentrations 1,000 times higher each test concentration group (0.40 mg/L group: 400 mg/L, 0.20 mg/L group: 200 mg/L, 0.10 mg/L group: 100 mg/L, 0.050 mg/L group: 50 mg/L, 0.0250 mg/L group: 25.0 mg/L).
Test solution was prepared by mixing the stock solution and dilution water in a constant flow rate (stock solution: 0.050 mL/min. dilution water: 50 mL/min.) The mixed test solutions were divided in four using splitter cells and sent to the individual testing containers.
- Controls: dilution water: Dechlorinated tap water (removed residual chlorine, etc from tap water supplied by Kurume city by processing with activated carbon, and it was sufficiently aerated) was used. As main water quality parameters of dilution water, hardness was 35.5 mg/L (CaCO3 equivalent), pH was 6.9. The residual chlorine was confirmed once a day to be not higher than 0.02 mg/L by ortho-tolidine method.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnia magna
- Strain/clone: not specified
- Source: This species was originally obtained from U.S. EPA Environmental Research Laboratory, Duluth, and it has been bred and raised in this laboratory.
- Age of parental stock (mean and range, SD): Daphnia, which have been sub-cultured, aged 2 to 4 weeks old were provided as the parent of test daphnia. When they matured and started to produce juveniles, the juveniles were removed at least at the day before the test. However, the daphnia from the batches with large number of dead individuals and batches producing resting eggs and males were not used.
- Feeding during test: continuously given by using metering pump to keep the concentration of Chlorella vulgaris at 2 - 4 x 10E8 cells/L.

ACCLIMATION: not needed since breeding water and test water were the same.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Juveniles of daphnia magna aged 24-hour or less were used for this test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

35.5 mg/L CaCO3 (below the recommended value of 140 mg/L, but validiaty criteria met, thus this low water hardness was not a concern).

Test temperature:

19.5-21.0°C, hence it was in the range of 20±1°C.

pH:

7.33-7.84, hence it varied 1 or less.

Dissolved oxygen:

7.9-9.0 mg/L, hence it was 60% or more of saturated dissolved oxygen concentration in all test vessels (saturated dissolved oxygen concentration at 20.0°C: 8.84 mg/L).

Nominal and measured concentrations:

Nominal: 0.0250-0.050-0.10-0.20-0.40 mg/L
Mean measured: 0.0218-0.046-0.0955-0.172-0.395 mg/L
Measured values of test substance concentration during exposure period were 0.0202 – 0.454 mg/L (nominal value 0.0250 – 0.40 mg/L), which were in the range 74.1 – 120% of the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel / Material, size, headspace, fill volume:3.0 L glass container (16cm in diameter, 17cm in depth). Water discharge holes were placed in the sides of the containers in order to maintain a volume of approximately 1.8 L.
- Aeration: not specified
- Type of flow-through (e.g. peristaltic or proportional diluter): A diluter which continuously supplies test water diluted to the required concentration levels from stock solution was used. Metering pump: Minichemical pump SP-D-2500(S) type (NIHON SEIMITSU KAGAKU CO., LTD.)
- Renewal rate of test solution (frequency/flow rate): about 10 times/day
- No. of organisms per vessel: 10 animals per replicate: hence, 40 animals per test group
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates
- Biomass loading rate: 10 dahnia in 1.8 L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water (removed residual chlorine, etc from tap water supplied by Kurume city by processing with activated carbon, and it was sufficiently aerated) was used. As main water quality parameters of dilution water, hardness was 35.5 mg/L (CaCO3 equivalent), pH was 6.9. The residual chlorine was confirmed once a day to be not higher than 0.02 mg/L by ortho-tolidine method.
- Total organic carbon: COD: 0.2 mg/L
- Particulate matter: not specified
- Metals: not detected
- Pesticides: not detected
- Chlorine: not detected
- Alkalinity: 21.0 mg/L
- Ca/mg ratio: not specified
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, pH and disolved oxygen measured at 0, 3, 10, 14, 17 and 21-day.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light / 8-hour dark
- Light intensity: Room light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Death number and mortality rate of parental daphnia, First brood production date, Mean cumulative number of produced juveniles, Size and condition of parental daphnia, Occurrence of resting-eggs

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: na
- Range finding study: Based on the 48-h EiC50 value (0.447 mg/L) obtained from acute immobilisation test, 5 concentration groups (0.40, 0.20, 0.10, 0.050 and 0.0250 mg/L) were established as test concentrations.

Reference substance (positive control):

not specified

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.116 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI: 0.0991-0.137 mg/L

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.131 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CI: 0.112-0.157 mg/L

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: not quantitatively performed
- Other biological observations: In the groups of 0.0250 and 0.050 mg/L and control group, no abnormal symptom was observed during exposure period.
In the group of 0.10 mg/L, increased brightness of body color began from day 12, and small size individuals have begun to be seen from day around 15.
In the group of 0.20 mg/L, symptom of immobilization has begun to be seen in some individuals from day around 2. Thereafter, increased brightness of body color and small size individuals have begun to be seen, and from around 1 week, about half number of the individuals became small in size.
In the concentration group of 0.40 mg/L, the highest concentration of the test, symptom of immobilization has begun to be seen in some individuals from day around 2 after the beginning of exposure, and thereafter, they became increased brightness of body color and lethargic state. Small size individuals of daphnia compared with control group have begun to appear from day 4.
Occurrence of resting-eggs, etc.: During exposure period, there was no occurrence of resting-eggs in any test groups. Though falling-eggs were observed in each concentration group and control group, except for the group of 0.40 mg/L, large difference was not observed among the test groups. Besides, dead individuals were rarely seen in produced juveniles.

- Mortality of control: 3 parent animals were dead at the end of the 21d exposure
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Effect concentrations exceeding solubility of substance in test medium: none reported

Results with reference substance (positive control):

Test with reference substance was not performed in this study report.

Death number and mortality rate of parental daphnia:

Cumulative mortality of parental daphnia in control group was 7.5% at the end of exposure, hence, it satisfied the criterion of 20% or less which is the condition of the validity of test.

At the concentration groups of 0.10 mg/L or lower, cumulative mortalities were 20% or less at the end of exposure, hence, there was no statistical significant difference compared with control group.

At the concentration groups of 0.20 and 0.40 mg/L, cumulative mortalities were 65% and 100% respectively, therefore, apparent effect was found on survival, besides, statistical significant difference was suggested when compared with control group.

[p=0.05, statistical method: Kruskal-Wallis’ rank-sum test and Non-parametricmultiple comparison technique Dunnett method]

First brood production date:

The first brood production date of parental daphnia in control group was day 7, hence, it satisfied the criterion of day 9 or less which is the condition of the validity of test.

At the concentration groups of 0.20 mg/L or lower, the first brood production dates of parental daphnia were day 7: it was the same as control group.  At the concentration group of 0.40 mg/L, no production of juveniles appeared during exposure period.

Mean cumulative number of produced juveniles:

In control group, parental daphnia produced 5-6 of broods, and the mean cumulative number of produced juveniles per parental 1 daphnia was 333 animals, hence, it satisfied the criteria of 3 broods or more, 20 animals which are the conditions of the validity of test.

At the concentration groups of 0.0250 and 0.050 mg/L, parental daphnia produced 5-6 of broods and the mean cumulative numbers of produced juveniles per parental 1 daphnia were 349 animals and 325 animals respectively, hence, it suggested the similar fecundity status compared with control group.

At the concentration groups of 0.10 mg/L or higher, the mean cumulative numbers of produced juveniles were 152 animals or less per parental 1 daphnia, hence, it was significantly small when compared with control group.

[p=0.05, statistical method: one-way analysis of variance and Dunnett’s multiple comparison technique]

Validity criteria fulfilled:

yes

Remarks:

Mortality of the parent animals did not exceed 20% at the end of the test; the mean number of living offspring produced per parent animal surviving at the end of the test is >= 60.

Conclusions:

21d-LC50 = 0.131 mg/L (95% confident interval: 0.112 – 0.157 mg/L)
21d-EC50% Reproduction Inhibition = 0.116 mg/L (95% confident interval: 0.0991 – 0.137 mg/L)
21d-NOECreproduction = 0.050 mg/L
21d-LOECreproduction = 0.10 mg/L
All of the above concentrations are the values based on nominal values

Executive summary:

The purpose of the study was to determine the long term effect over 21 days of the test item on Daphnia magna, according to the OECD 202 guideline and following GLP. Forty daphnids (10 animals per replicate, 4 replicates), were used per concentration level and control. At test start the daphnids were ared 24 hours old or less. The study was carried out under flow-through conditions. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the first appearance of living juveniles, occurrence of resting eggs, adult mortality, size and conditions of parental animals. Nominal concentrations of the test item were as follows: 0.40 – 0.20 – 0.10 – 0.050 – 0.0250 mg/L. The test item concentrations were analytically verified via HPLC (High Performance Liquid Chromatography) on days 0, 3, 7, 14 and 21. The measured values of test substance concentration during exposure period were 0.0202 – 0.454 mg/L (nominal value 0.0250 – 0.40 mg/L), which were in the range 74.1 – 120% of the nominal concentrations. The environmental conditions were within the acceptable limits. The validity criteria of the OECD 202 test guideline were met. Based on the above results chemical consider to be classified as aquatic acute 1/ chronic 1 as per the CLP classification criteria.

21d-LC50 = 0.131 mg/L (95% confident interval: 0.112 – 0.157 mg/L)

21d-EC50% Reproduction Inhibition = 0.116 mg/L (95% confident interval: 0.0991 – 0.137 mg/L)

21d-NOECreproduction = 0.050 mg/L

21d-LOECreproduction = 0.10 mg/L

All of the above concentrations are the values based on nominal values.

Description of key information

21d-LC50 = 0.131 mg/L (95% confident interval: 0.112 – 0.157 mg/L)

21d-EC50% Reproduction Inhibition = 0.116 mg/L (95% confident interval: 0.0991 – 0.137 mg/L)

21d-NOECreproduction = 0.050 mg/L

21d-LOECreproduction = 0.10 mg/L

All of the above concentrations are the values based on nominal values

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.05 mg/L

Additional information

The purpose of the study was to determine the long term effect over 21 days of the test item on Daphnia magna, according to the OECD 202 guideline (1984) and following GLP. Forty daphnids (10 animals per replicate, 4 replicates), were used per concentration level and control. At test start the daphnids were ared 24 hours old or less. The study was carried out under flow-through conditions. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the first appearance of living juveniles, occurrence of resting eggs, adult mortality, size and conditions of parental animals. Nominal concentrations of the test item were as follows: 0.40 – 0.20 – 0.10 – 0.050 – 0.0250 mg/L. The test item concentrations were analytically verified via HPLC (High Performance Liquid Chromatography) on days 0, 3, 7, 14 and 21. The measured values of test substance concentration during exposure period were 0.0202 – 0.454 mg/L (nominal value 0.0250 – 0.40 mg/L), which were in the range 74.1 – 120% of the nominal concentrations. The environmental conditions were within the acceptable limits. The validity criteria of the OECD 202 test guideline were met. According to the effect chemical was consider as toxic and classified as aquatic acute 1/ chronic 1 category as per the CLP classification criteria.

21d-LC50 = 0.131 mg/L (95% confident interval: 0.112 – 0.157 mg/L)

21d-EC50% Reproduction Inhibition = 0.116 mg/L (95% confident interval: 0.0991 – 0.137 mg/L)

21d-NOECreproduction = 0.050 mg/L

21d-LOECreproduction = 0.10 mg/L

All of the above concentrations are the values based on nominal values.

Long term toxicity study of test chemical were reviewed by test conducting according to OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test and Reproduction Test). Test conducted under the flow-through method. Chemical analytically monitorized by HPLC with the frequency of 0th, 3rd, 7th, 14th, 21st day. 40 per concentration (10 per vessel) daphnia were incorporated. The effective concentration (EC50) value of test chemical based on the effect on reproduction in Daphnia magna in a 21 day study was determine to be 0.116 mg/L and the NOEC value was observed to be 0.05 mg/L. Thus based on the long term effects, it was concluded that the chemical was toxic and classified as aquatic chronic 2 category as per the CLP classification criteria.

Thus chemical consider to be toxic