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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 May 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 201 guideline and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Version March 23, 2006 (Annex 5 Correction July 28, 2011).
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
Remarks:
high performance liquid chromatography with dual mass spectrometry (LC-MS/MS)
Details on sampling:
Concentrations: 5 concentrations were tested and analysed
- Sampling frequency : at initiation (0 hour), 24, 48,and 72 hours (termination). Time 0-hour samples were collected from parent solutions. Samples at 24 and 48 hours were collected from analytical replicates. Samples at 72 hours were collected after combining replicate solutions (A through F for the control and A through C for test substance treatments).
- Sampling method: 10-mL sample was collected, centrifuged for 10 minutes at 5,000 RPM and a 5-mL aliquot of the supernatant transferred to a new culture tube. The samples were then diluted to a volume of 10 mL with ACN and further diluted, if necessary, with 50:50 ACN:HPLC water to provide final concentrations within the standard concentration range (1.00 to 10.0 ng a.i./mL).
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 LC-MS/MS.
- Sample storage conditions before analysis: not reported

Vehicle:
no
Details on test solutions:
The nominal concentrations are : 0 (control), 0.029, 0.092, 0.29, 0.94, and 3.0 mg a.i./L. A primary standard was prepared by transferring 0.0101 g (0.0100 g corrected for purity) of A017 to a 1000-mL glass volumetric flask and bringing the flask to volume with test medium for a concentration of 0.010 mg a.i./mL. The primary standard solution was sonicated for approximately 10 minutes following preparation and was clear and colorless with no undissolved test substance. Appropriate aliquots of the primary standard were diluted to a volume of 1.0 L with test medium to prepare the test substance treatments at concentrations of 0.029, 0.092, 0.29, 0.94, and 3.0 mg a.i./L. The control consisted of test medium only.

Preparation of analytical standards and Matrix Spiking Solutions :
A primary stock solution of A017 was prepared 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 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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata
- Reason for the selection of the test organism : Pseudokirchneriella subcapitata is a suitable green algae species according to the guideline
- Source : Department of Botany, Culture Collection of Algae, University of Texas at Austin,
- Age of inoculum (at test initiation): three days old and the biomass had increased exponentially (i.e., specific growth rate of 1.3 day-1) during the culture period.
- Method of cultivation: Periodically, new cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those used for testing (temperature-controlled (24 ± 2° C) environmental chamber under continuous light (~8,600 lux)).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
Required : 21-24°C, controlled at ± 2°C during the study period
Measured during the test : 23.1°C to 24°C
pH:
Required : pH fin d'essai témoin = pH initial ± 1.5
Measured during the test : 7.3 to 8.1
Dissolved oxygen:
not reported
Salinity:
not applicable
Conductivity:
not reported
Nominal and measured concentrations:
Nominal Concentrations: 0 (control), 0.029, 0.092, 0.29, 0.94, and 3.0 mg a.i./L
72-Hour Geometric Mean Measured Concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml Erlenmeyer flasks
- Type (delete if not applicable): closed with foam stoppers
- solutions volume: 100 ml
- Aeration: The flasks were swirled on an orbital shaker table at 100 rpm throughout the test. Oscillation rate was recorded daily during the definitive exposure
- Initial cells density: algal concentrate containing approximately 4.9 E+5 cells/mL, resulting in a final density of approximately 5.0 E+3 cells/mL for each flask
- Control end cells density: 187 E+4 cells/mL
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates
- 1 additional replicate with 0.029 mg a.i./L test substance treatment, containing 100 mL of the appropriate parent solution but no algae.
- Two additional replicates for the control and all test substance treatments, containing 100 mL of the appropriate parent solution and algae were also prepared and used for analytical evaluations at 24 and 48 hours.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
The test medium was a freshwater algal nutrient medium . The medium was prepared by the addition of appropriate reagent grade salts to autoclaved reagent water. Reagent water is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, de-mineralization, and organic adsorption cartridges, and then through a 0.2-μm filter. After preparation, the medium was filtered through 0.2-μm Millipore® filters.
Tha ABC water is analysed regularly for metals, chlorinated pesticides, Polychlorinated Biphenyls (PCBS), Chlorinated Herbicides, Organophosphorus Pesticides, Chlorine and Nitrogen (Ammonia, nitrate, nitrite) and Phosphorus total.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24h light
- Light intensity and quality: 7,713 to 7,863 lux, (measureddaily with a LI-COR Model LI-250A light meter)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: direct microscopic counting with a hemacytometer

PHYSICO-CHEMICAL PARAMETERS MEASURED
-pH value measured at the beginning of exposure in additionnal replicate of each concentration and the control, and at the end of exposure in a pooled sample of each concentration and the control with a WTW Model pH 330i pH meter
- Room temperature : A continuous recording of environmental chamber temperature was made from one uninoculated blank flask using an electronic datalogger with thermistor probe.
- Light intensity measured daily with a LI-COR Model LI-250A light meter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Initial range-finding test : 0 (control), 0.10, 1.0, 5.0, 10, and 100 mg a.i./L substance treatments performed  and  the cell density was 55.8 E+4 cells/ml (control) and 42.9; 25.4; 0.389 and 0.278 E+4 cells/ml respectively.
- Second range-finding test : 0 (control), 0.0030, 0.030, 0.30 and 3.00 mg a.i./L substance treatments performed  and  the cell density was 64.9 E+4 cells/mL (control) and 79.6; 74.0; 62.1 and 5.72 E+4 cells/mL respectively.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding tests, a nominal concentration range of 0 (control), 0.029, 0.092, 0.29, 0.94, and 3.0 mg a.i./L was selected for the definitive test.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 2.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 2.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.017 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.162 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limit : 0.0232 - 0.301 mg ai/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.017 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no

Concentrations of A017 were not maintained within 20% of the nominal concentrations during the exposure. Therefore the biological response results are reported based upon 72-hour geometric mean concentrations. The geometric mean calculated concentrations in the test substance treatment solutions after 72 hours were 0.0173, 0.0674, 0.227, 0.723, and 2.18 mg a.i./L, which represented recoveries of 60 to 78% of the nominal concentrations.

Percent inhibition in growth rate from time zero to 72 hours, as compared to the control, ranged from 0% at the concentration of 0.0173 mg a.i./L to 23% at the concentration of 2.18 mg a.i./L. The NOEC at 72 hours was determined to be 0.0173 mg ai/L. The coefficient of variation of average specific growth rates during the whole test period in control replicates was 0%. The mean coefficient of variation in growth rate between adjacent time periods was 18% for the control replicates.

Percent inhibition in yield from time zero to 72 hours, as compared to the control, ranged from -2% at the concentration of 0.0173 mg a.i./L to 75% at the concentration of 2.18 mg a.i./L. The NOEC at 72 hours was determined to be the 0.0173 mg a.i./L.


Reported statistics and error estimates:
The NOEC values, based on growth rate from time zero and yield, were estimated using a oneway analysis of variance (ANOVA) procedure and a one-tailed Dunnett’s test (p = 0.05) where the alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control. Prior to the Dunnett’s test, a Shapiro-Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk’s and Levene’s tests indicated normality and insignificant heterogeneity (i.e., p > 0.01), the analysis was performed on the non-transformed raw data. In instances of non-normality or heterogeneity (i.e., p < 0.01), a square root transformation was performed. If both the non-transformed raw data and the transformed data exhibited non-normality or inequality of variance, a non-parametric analysis of variance was performed on the ranks of the raw data values. Parametric analyses were performed on the 0-24 hour growth rate and 24 hour yield data. Non-parametric analyses were performed on the 0-48 and 0-72 hour growth rate and the 48 and 72 hour yield data.

Measured Concentrations of A017 During the 72-Hour Growth Inhibition Test with the Unicellular Green Alga, Pseudokirchneriella subcapitata

NominalConc (mga.i./L)

Meas. Conc. mg a.i. /L (Percent nominal)

24h-geometric

Mean (mg a.i./L)

48h-geometric

Mean (mg a.i./L)

72h-geometric

Mean (mg a.i./L)

0-hour

24-hour

48-hour

72-hour

Control

<MLQa

<MLQa

<MLQa

<MLQa

<MLQa

<MLQa

<MLQa

0.029

0.0280 (97)

0.0226

(78)

0.0175

(60)

0.00812

(28)b

0.0252

(87)

0.0223

(77)

0.0173

(60)

0.029 (abiot)

-

0.0191

(66)

0.0120

(41)

0.0124

(43)

-

-

-

0.092

0.0924 (100)

0.0720

(78)

0.0650

(71)

0.0477

(52)

0.0816

(89)

0.0756

(82)

0.0674

(73)

0.29

0.294

(101)

0.227

(78)

0.237

(82)

0.167

(58)

0.258

(89)

0.251

(87)

0.227

(78)

0.94

0.902

(96)

0.781

(83)

0.624

(66)

0.621

(66)

0.839

(89)

0.760

(81)

0.723

(77)

3.0

2.92

(97)

2.23

(74)

2.13

(71)

1.64

(55)

2.55

(85)

2.4

(80)

2.18

(73)

(a)    Minimum Quantifiable Limit (MQL) = 0.00200 mga.i./L

(b)     Re-analyzed in duplicate, mean of duplicate re-analyses reported.

 

 

Growth Rate Values From Time Zero for a Pseudokirchneriella subcapitata during a 72 -Hour Exposiure to A017

72h Geom. Mean Conc. (mga.i./L)

Mean Growth Rate (cells/ml/hour)

%inhibitionb

0-24 hours

0-48hours

0-72 hours (% CV)

0-72 hours

Control

0.0850

0.0901

0.0823 (CV: 0)

-

0.0173

0.0858

0.0897

0.0825 (CV: 0)

0

0.0674

0.0858

0.0875*

0.0696* (CV: 2)

15

0.227

0.0852

0.0832*

0.0699* (CV: 0)

15

0.723

0.0799

0.0787*

0.0697* (CV: 0)

15

2.18

0.0730*

0.0686*

0.0634* (CV: 1)

23

(a)    Mean growth rate values calculated using the individual replicate growth rates. Values are rounded to three significant figures.

(b)    Percent inhibition as compared to the control was determined at 72 hours using the following equation:

% inhibition = ([( mean control growth rate) - (mean treatment growth rate)] / (mean control growth rate) ) x 100

* Significant reduction in growth rate as compared to the control (Dunnett’s test, p= 0.05).

 

 

Yield Values for Pseudokirchneriella subcapitata During a 72-Hour Exposure to A017

72h Geom. Mean Conc. (mga.i./L)

Yield (cells/ml x 104)a

%inhibitionb

24 hours

48hours

72 hours (% CV)

72 hours

Control

3.35

37.5

187 (CV: 2)

-

0.0173

3.43

36.5

190 (CV:1)

-2

0.0674

3.43

32.9*

74.7* (CV:7)

60

0.227

3.39

26.7*

76.4* (CV:1)

59

0.723

2.91

21.4*

75.2* (CV:1)

60

2.18

2.39*

12.9*

47.6* (CV:4)

75

 (a)    Mean yield calculated using the individual replicate yield. Yield is claculated as the biomass at the end of the exposure period minus the starting biomass (i.e., target inoculation cell density of 5.0 E+3 cells/mL). Values are rounded to three significant figures.

(b)     Percent inhibition as compared to the control was determined at 72 hours using the following equation:

% inhibition = ([( mean control yield) - (mean treatment yield)] / (mean control yield)) x 100

(*)   Significant reduction in yield as compared to the control (Dunnett’s test, p= 0.05).

Validity criteria fulfilled:
yes
Conclusions:
The test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata with the following effect values expressed as geometric mean measured concentrations. The EC50 values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours of exposure were >2.18 mg a.i./L and 0.162 (0.0232 - 0.301) mg a.i./L respectively. The ErC10 and NOEC values for inhibition of specific growth were > 2.18 and 0.0173 mg a.i./L respectively, and the EyrC10 and NOEC values for yield were <0.0173 and 0.0173 mg a.i./L respectively. The most reliable value is considered to be 72h-ErC10 growth rate > 2.18 mg a.i./L (geometric mean measured concentration).
Executive summary:

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was tested according to the OECD guideline 201 and GLP.

The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions. The algae at the initial concentration of 5 x 103cells/ml were exposed during 72 hours to the following nominal concentrations 0 (control), 0.029, 0.092, 0.29, 0.94, and 3.0 mg a.i./L. The tested concentrations, analysed by High Performance Liquid Chromatography with dual mass spectrometry (LC-MS/MS), were not maintained within 80% of the nominal concentrations during the exposure. Therefore the biological response results are reported based upon the following geometric mean concentrations : 0.0173, 0.0674, 0.227, 0.723, and 2.18 mg a.i./L, which represent recoveries of 60 to 78% of the nominal concentrations.

All the validity criteria of the guidelines were fulfilled : the pH did not increase more than 1.5 units, the biomass in the controls increased 374 times, the coefficient of variation of average specific growth rates during the whole test period in control replicates was 0% and the mean coefficient of variation in growth rate between adjacent time periods was 18%.

The EC50 values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours of exposure were >2.18 mg a.i./L and 0.162  (0.0232 - 0.301) mg a.i./L respectively. The ErC10 and NOEC values for inhibition of specific growth were > 2.18 and 0.0173 mg a.i./L respectively, and the EyrC10 and NOEC values for yield were <0.0173 and 0.0173 mg a.i./L respectively.

Since the validity criteria are fulfilled for the growth rates in the controls, the results of the inhibition of the test item on the growth rate are prefered to the results obtained on the yield. The most reliable value to expressed the inhibition of the test item to the green algae Pseudokirchneriella subcapitata is considered to be 72h-ErC10 growth rate > 2.18 mg a.i./L (geometric mean measured concentration).

Description of key information

The test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata with the following effect values expressed as geometric mean measured concentrations. The EC50 values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours of exposure were >2.18 mg a.i./L and 0.162  (0.0232 - 0.301) mg a.i./L respectively. The ErC10 and NOEC values for inhibition of specific growth were > 2.18 and 0.0173 mg a.i./L respectively, and the EyrC10 and NOEC values for yield were <0.0173 and 0.0173 mg a.i./L respectively. The most reliable value is considered to be 72h-ErC10 growth rate > 2.18 mg a.i./L (geometric mean measured concentration).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
2.18 mg/L

Additional information

The toxicity of the test item to the green algaePseudokirchneriella subcapitatawas tested according to the OECD guideline 201 and GLP.

The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions. The algae at the initial concentration of 5 x 103cells/ml were exposed during the 72 hours to the following nominal concentrations 0 (control), 0.029, 0.092, 0.29, 0.94, and 3.0 mg a.i./L. The tested concentrations, analysed by High Performance Liquid Chromatography with dual mass spectrometry (LC-MS/MS), were not maintained within 80% of the nominal concentrations during the exposure. Therefore the biological response results are reported based upon the following geometric mean concentrations : 0.0173, 0.0674, 0.227, 0.723, and 2.18 mg a.i./L, which represent recoveries of 60 to 78% of the nominal concentrations.

All the validity criteria of the guidelines were fulfilled : the pH did not increase more than 1.5 units, the biomass in the controls increased 374 times, the coefficient of variation of average specific growth rates during the whole test period in control replicates was 0% and the mean coefficient of variation in growth rate between adjacent time periods was 18%.

The EC50 values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours of exposure were >2.18 mg a.i./L and 0.162  (0.0232 - 0.301) mg a.i./L respectively. The ErC10 and NOEC values for inhibition of specific growth were > 2.18 and 0.0173 mg a.i./L respectively, and the EyrC10 and NOEC values for yield were <0.0173 and 0.0173 mg a.i./L respectively.

Since the validity criteria are fulfilled for the growth rates in the controls, the results of the inhibition of the test item on the growth rate are prefered to the results obtained on the yield. The most reliable value to express the inhibition of the test item to the green algae Pseudokirchneriella subcapitata is considered to be 72h-ErC10 growth rate > 2.18 mg a.i./L (geometric mean measured concentration).