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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 June 2016 to 29 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Test performed according to OECD 202 guideline and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Version April 13, 2004.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Name: A017, 1-Naphthol
Batch Number: ADT1214003
CAS Number: 90-15-3
Storage Conditions: Ambient
Initial analysis : 27 May 2016 and purity : 98.9%
Re-analysis Date: 23 September 2016 and purity 99.2%
The purity is unchanged and remain 98.9%
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 5 concentrations were tested and analysed.
- Sampling method: The concentrations of A017 were measured in samples collected from the control and each test substance treatment. Samples from freshly-prepared parent solutions were collected at initiation (0 hour) and composite samples from replicates A through D from the control and each test substance treatment were collected at 48-hours (termination). A017-fortified samples were also prepared for analysis at each sample period.
- Sample storage conditions before analysis: At each sampling time point, a 5 mL sample was collected from the control and each test substance treatment and placed into separate 10-mL culture tubes. The samples were diluted with 5 mL of ACN and further diluted, if necessary, with 50:50 ACN:HPLC water to provide final concentrations within the analytical standard concentration range (0.0500 to 2.00 ng A017/L). Six samples (three low spike and three high spike) were prepared at each of two quality control (QC) fortification concentrations that bracket the expected high and low test substance treatment concentrations and analyzed in a similar manner. The samples were vialed and analyzed by HPLC-UV.
Vehicle:
no
Details on test solutions:
The following nominal concentrations of 0 (control), 0.40, 0.80, 1.6, 3.2, and 6.4 mg A017/L were tested. A 0.0128 mg A017/mL primary standard was prepared at test initiation by diluting 0.0129 g A017 (0.0128 g adjusted for 98.9% purity) with dilution water to a volume of 1000 mL. Test solutions were prepared by diluting appropriate aliquots of the primary standard to a volume of 1.0 L with dilution water. The control consisted of dilution water only.
In the range finding study (from 0 to 100 mg/l ) the 0 (control) and all test substance treatments were clear and colorless with no visible particulates, surface film, undissolved test substance, or precipitate throughout the test.

Preparation of analytical standards and Matrix Spiking Solutions :
A primary stock solution of A017 was prepared on 27 June 2016 at a concentration of 0.665 mg A017/mL by weighing 16.8 mg of A017 into a 25-mL volumetric flask, correcting for 98.9% purity, and bringing the flask to volume with acetonitrile (ACN). Subsequent dilutions of this solution in 50:50 HPLC water:ACN were used as analytical standards during the definitive test.
A second primary stock solution of A017 was prepared on 27 June 2016 at a concentration of 3.91 mg A017/mL by weighing 98.9 mg of A017 into a 25-mL volumetric flask, correcting for 98.9% purity, and bringing the flask to volume with ACN. Subsequent dilutions of this solution in ACN were used to prepare QC fortifications during the definitive test.
All solutions were stored refrigerated when not in use.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna neonates (<24-hours old)
- Source: an in-house daphnid culture
- Age of parental stock (mean and range, SD): Neonates for the definitive test were collected from a single culture containing adults that were approximately 31 days old
- Feeding during test : No

ACCLIMATION
- Acclimation period: Since the culturing and testing environmental parameters were equivalent (i.e. temperature, dilution water, and lighting), no acclimation period was necessary
- Acclimation conditions : same as test
- Type and amount of food: suspension of the algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) supplemented by an artificial diet consisting of a wheat grass, salmon starter, and yeast suspension.
- Feeding frequency: at least once a day
- Health during acclimation (any mortality observed): The adults were considered acceptable with no signs of stress, disease or physical damage.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Five neonates (<24-hours old) were added to each of four test chambers per treatment at the start of the test. Daphnids were impartially added to a set of labeled containers with each container representing one treatment replicate. Each container was then randomly assigned to a treatment replicate by random number generator. The individuals within each container were then transferred into the corresponding test chamber below the surface of the solution using a wide bore pipet.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The total alkalinity and hardness of the dilution water at test initiation were 162 mg CaCO3/L and 148 mg CaCO3/L, respectively.
Total hardness and alkalinity were measured in the dilution water at test initiation using titrimetric methods.
Test temperature:
Test solution temperatures during the 48-hour exposure ranged from 19.7 to 20.6°C as measured in the individual test replicates. The temperature range remained within 20 ± 1°C.
Temperature was measured at 0 hour (initiation) and 48 hours (termination) in all replicates with a WTW Multi Meter 343.
A thermistor probe was positioned in a surrogate test chamber in the water bath to continuously record temperature.
pH:
Test solution pH ranged from 8.5 to 8.6 throughout the test.
pH was measured at 0 hour (initiation) and 48 hours (termination) in all replicates with a WTW Multi Meter 343.
Dissolved oxygen:
Dissolved oxygen concentrations ranged from 8.6 to 8.9 mg/L (101 to 102% saturation) throughout the test.
Dissolved oxygen concentrations was measured at 0 hour (initiation) and 48 hours (termination) in all replicates with a WTW Multi Meter 343.
Conductivity:
The conductivity of the dilution water at test initiation was 344 μS/cm.
The conductivity was measured in the dilution water at test initiation with a WTW Cond 3300i conductivity meter.
Nominal and measured concentrations:
Measured concentrations of A017 in the test substance treatment solutions at initiation were 0.480, 0.740, 1.69, 3.35, and 6.65 mg A017/L, which represented recoveries of 93 to 120% of the nominal concentrations. Measured concentrations of A017 in 48 hours (termination) were 0.452, 0.845, 1.66, 3.18, and 6.36 mg A017/L, which represented recoveries of 99 to 113% of the nominal concentrations. The mean measured concentrations in the test substance treatment solutions during the 48-hour exposure were 0.466, 0.793, 1.68, 3.27, and 6.51 mg A017/L, which represented recoveries of 99 to 117% of the nominal concentrations. No residues of A017 were detected in the control solutions above the MQL of 0.100 mg A017/L. Recoveries from QC fortifications ranged from 100 to 116% of the nominal concentrations .
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass jars containing 200 ml and covered with a clear plastic lid
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): No renewal (static test)
- No. of organisms per vessel: 5 neonates (< 24 hours old) added to each of 4 test chambers
- No. of vessels per concentration (replicates): 4 vessels per concentrations
- No. of vessels per control (replicates):4 vessels
- No. of vessels per vehicle control (replicates): no vehicule
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO). The well water and RO water were blended together to yield a total hardness of 130 to 160 mg CaCO3/L. Prior to use, the dilution water was passed through a sediment filter. Characterization of the base water, i.e., well water, used to prepare the dilution water is reported in the annexe of the report
- Total organic carbon: less than 2.0 mg/L
- Metals, chlorinated pesticides, Polychlorinated Biphenyls (PCBS), Chlorinated Herbicides, Organophosphorus Pesticides, Chlorine and Nitrogen (Ammonia, nitrate, nitrite) and Phosphorus total were checked.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Fluorescent lighting was maintained on a 16-hour daylight photoperiod with 30-minute simulated dawn and dusk periods.
- Light intensity: The measured light intensity at the start of the definitive test was 569 lux as measured with a LI-COR Model LI-250A light meter equipped with a photometric sensor.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The daphnids were observed for immobility and sublethal effects at 24 and 48 hours (±1 hour) after test initiation

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 0.010, 0.10, 1.0, 5.0, 10, and 100 mg A017/L substance treatments performed and 0, 0, 0, 0, 100, 100, and 100% immobility observed respectively.
- Results used to determine the conditions for the definitive study: at 1 mg/L no immobilisation was observed and at 5 mg/L there were 100% immobilisation. The tested concentrations in the definitive teste were between 0.4 mg/L and 6.4 mg/L
Reference substance (positive control):
not required
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 6.51 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.51 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence limit : 2.29 - 2.75 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.68 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: Lack of statistically significant immobility and sublethal effects.
Details on results:
- Behavioural abnormalities:
- Other biological observations: not reported
- Mortality of control: 5%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no difference between nominal and measured concentrations
- Effect concentrations exceeding solubility of substance in test medium: the solutions were clear and colorless with no visible particulates, surface film, undissolved test substance, or precipitate throughout the test until 100 mg/L (range finding test). All daphnids appeared normal with no behavioral or morphological abnormalities.
Results with reference substance (positive control):
Not available
Reported statistics and error estimates:
All statistical analyses were performed with SAS software (version 9.3). Estimates of EC50 values and their 95% confidence limits were calculated using the probit . When the P value for Goodness of Fit was >0.05 and there was no other evidence of questionable convergence, the probit method was selected for reporting. The no-observed-effect concentration (NOEC) was determined using a Fisher’s exact test.

Measured Concentrations of A017 During the 48-Hour Static, Acute Toxicity

Test with Daphniamagna

Nominal

Concentration

(mg A017/L)

Measured Concentration as mg A017/L(Percent Nominal)

 

0-Hour

48-Hour

MeanMeasured

Concentration

(mg A017/L)

Control

<MQL*

<MQL*

<MQL*

0.4

0.480 (120)

0.452 (113)

0.466 (117)

0.8

0.740 (93)

0.845 (106)

0.793 (99)

1.6

1.69 (106)

1.66 (104)

1.68 (105)

3.2

3.35 (105)

3.18 (99)

3.27 (102)

6.4

6.65 (104)

6.36 (99)

6.51 (102)

*MQL = Minimum Quantifiable Limit  

Immobility of Daphnia magna Exposed to A017 for 48 Hours Under Static Test Conditions

Mean Measured

Concentration

(mg A017/L)

R

E

P

Cumulative Percent Immobile

(%SublethalEffects)

24H

48H

TreatmentMean

Percent Immobile

(48Hours)

0 (Control)

A

20 (0)

20 (0)

5

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

0.466

A

0 (0)

0 (0)

0

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

0.793

A

0 (0)

0 (0)

0

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

1.68

A

0 (0)

0 (0)

0

B

0 (0)

0 (0)

C

0 (0)

0 (0)

D

0 (0)

0 (0)

3.27

A

40 (0)

100 (0)

90*

B

20 (0)

100 (0)

C

40 (0)

80 (0)

D

0 (0)

80 (0)

6.51

A

40 (0)

100 (0)

100*

B

40 (0)

100 (0)

C

40 (0)

100 (0)

D

20 (0)

100 (0)

Note: Five daphnids were placed in each replicate at test initiation, totaling 20 daphnids per treatment.

* Statistically significant reduction in survival as compared to the control (Fisher’s One-Tailed Exact Test, p < 0.05)

 

 

Validity criteria fulfilled:
yes
Remarks:
Control daphnids immobilisation was 5% (acceptability limit 10%) and dissolved oxygen concentration was ≥ 8.8 mg/L in control and test substance treatments (acceptability minimum of 3 mg/L)
Conclusions:
The EC50 on Daphnia magna after 48 h of exposure to the substance 1-naphtol (A017) under static conditions is 2.51 mg/L (95% confidence limit = 2.29 - 2.75 mg/L) expressed as mean measured concentration. The 48h-NOEC is 1.68 mg/L (mean measured concentration).
Executive summary:

The acute toxicity of the test item to Daphnia magna was tested according to the OECD guideline 202 and GLP. The study was conducted under static conditions. 20 daphnies were exposed during the 48-hour exposure to the following mean measured concentrations 0.466, 0.793, 1.68, 3.27, and 6.51 mg /L, determined by High Performance Liquid Chromatography with ultraviolet detection (HPLC-UV) system. These measured concentrations represent recoveries of 99 to 117% of the nominal concentrations.

The water quality parameter (pH, oxygen) measured at the start and at the end of exposure were within the acceptable limits. The validity criteria of the guideline were fulfilled.

The EC50 on Daphnia magna after 48 h of exposure to the test item is 2.51 mg/L (95% confidence limit = 2.29 - 2.75 mg/L) expressed as mean measured concentration. The 48h-NOEC is 1.68 mg/L (mean measured concentration).

Description of key information

The EC50 on Daphnia magna after 48 h of exposure to the substance 1-naphtol (A017) under static conditions is 2.51 mg/L (95% confidence limit : 2.29 - 2.75 mg/L) expressed as mean measured concentration. The 48h-NOEC is 1.68 mg/L (mean measured concentration).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.51 mg/L

Additional information

The acute toxicity of the test item to Daphnia magna was tested according to the OECD guideline 202 and GLP. The study was conducted under static conditions. 20 daphnies were exposed during the 48-hour exposure to the following mean measured concentrations 0.466, 0.793, 1.68, 3.27, and 6.51 mg /L, determined by High Performance Liquid Chromatography with ultraviolet detection (HPLC-UV) system. These measured concentrations represent recoveries of 99 to 117% of the nominal concentrations.

The water quality parameter (pH, oxygen) measured at the start and at the end of exposure were within the acceptable limits. The validity criteria of the guideline were fulfilled.

The EC50 on Daphnia magna after 48 h of exposure to the test item is 2.51 mg/L (95% confidence limit = 2.29 - 2.75 mg/L) expressed as mean measured concentration. The 48h-NOEC is 1.68 mg/L (mean measured concentration).