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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date: 30 August 2018 and Study completion date: 27 November 2018 and Definitive exposure dates: 23 to 2619 October 2018
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
Details on test material:
Name: XTJ-785, experimental
Lot No.: 9570-2-6738
CAS No.: Not listed
Purity: >92% C1214 alcohol, propoxylated, aminated, ethyoxylated
Expiry Date: No date established

Sampling and analysis

Analytical monitoring:
Details on sampling:
Sampling method:
- Number of samples taken from each solution: One sample at each interval. Additional samples of the test solution were also collected at each sampling interval and stored frozen as archives samples.
- Sampling location: Approximate midpoint from the surface, bottom, and sides of the vessel
- Sampling device: pipet
- Quality control (QC) samples: Three samples per sampling interval, prepared in dilution water at nominal concentrations approximating the test concentration range.
- Intervals of analytical verifications:
*0 hour: Exposure solutions in volumetric flasks prior to division into replicate vessels.
*72 hours: Composite of all replicates within each treatment and control; additional replicate for no algae solution.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Due to an expected low water solubility of the test substance, Water Accommodated Fractions (WAFs) were prepared individually. The primary stock solution (100 mg/L) was initially observed to be clear and colorless with visible, oily, particulates on the surface of the solution and visible undissolved test substance throughout the water column. The solution was covered with plastic wrap and allowed to mix slowly overnight using a magnetic stir plate and Teflon-coated stir bar. Following overnight mixing and an approximately 15-minute settling period, the solution was observed to be clear and colorless with visible, oily, particulates on the surface of the solution.
Exposure solutions were mixed for approximately one minute using a magnetic stir plate and Teflon-coated stir bar and were observed to be clear and colorless with no visible undissolved test substance following preparation. Control vessels were maintained under the same conditions as the treatment level solution.

Details are included in section on any other information on materials and methods incl. tables.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The water-accommodated fraction (WAF) was then siphoned from the beaker, avoiding the surface and bottom of the vessel, and was observed to be clear and colorless with no visible undissolved test substance.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Species: Pseudokirchneriella subcapitata
- Strain: 1648
- Class: Chlorophyceae
- Source: UTEX, The Culture Collection of Algae at the University of Texas, Austin, Texas (maintained in stock culture at Smithers Viscient)
- Age (Inoculum): Four days since previous transfer

- Culturing media: Algal Assay Procedure (AAP) medium, prepared with sterile, deionized source water
- Culture conditions (same as test or not): yes same as test.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

not available
Test temperature:
22 to 24 °C
not available
Dissolved oxygen:
not available
not applicable as fresh water was used.
not available
Nominal and measured concentrations:
- Nominal concentrations: 0.0020 / 0.0051 / 0.013 / 0.032 / 0.080 and 0.20% of a 100 mg/L water accommodated fraction (WAF)
- Geometric Mean Measured concentrations: 0.049 / 0.22 / 0.46 / 1.4 / 4.0 and 11 µg/L
Details on test conditions:
- Vessel size/Type: 250 mL glass flasks, fitted with stainless steel caps which permitted gas exchange.
- Vessel solution volume: 100 mL per replicate
- Vessel preparation: Cleaned according to the protocol prior to initiation, conditioned prior to use by rinsing with the appropriate exposure or control solution.
- Required starting cell density: Approximately 10 000 cells/mL
- Inoculum culture density: 1 621 000 cells/mL
- Inoculum volume added: 0.617 mL per vessel, added aseptically
- Initial (0-hour) cell density: 10 000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six
- Additional Replication: Replicate flask of the 0.013 of a 100 mg/L WAF (nominal) treatment level, not inoculated with algae, analyzed at 72 hours of exposure. Results of the solution without algae were compared with the results for the 0.013% of a 100 mg/L WAF solution contaning algae to estimate the impact that the presence of algal biomass had on the test substance concentration.

- Standard medium used: Algal Assay Procedure (AAP) Medium
- Detailed composition medium was used:

Composition of Algal Assay Procedure (AAP) Medium
Compound Concentration
NaNO3 25.5 mg/L
MgCl2•6H2O 12.16 mg/L
CaCl2•2H2O 4.41 mg/L
MgSO4•7H2O 14.7 mg/L
K2HPO4•3H2O 1.368 mg/L
NaHCO3 15.0 mg/L
H3BO3 185.5 µg/L
Na2SeO4a 1.88 µg/L
MnCl2•4H2O 415.4 µg/L
ZnCl2 3.270 µg/L
CoCl2•6H2O 1.43 µg/L
CuCl2•2H2O 0.012 µg/L
Na2MoO4•2H2O 7.26 µg/L
FeCl3•6H2O 160 µg/L
Na2EDTA•2H2O 300 µg/L

TEST MEDIUM / WATER PARAMETERS - Source/preparation of dilution water: Dilution water is AAP (Algal Assay Procedure) medium prepared with sterile deionized source water.
- Water Type (Testing): AAP medium
- Dilution water source (culture and testing): Town of Wareham water
- Total Organic Carbone: 0.39 mg/L

- Adjustment of pH: pH is 7.5 +/- 0.1, initial pH adjusted with dilute hydrochloric acid (HCl) or sodium hydroxide (NaOH) prior to use if necessary.
- Photoperiod: None (continuous)
- Light intensity and quality: 60 to 120 µE/m2/S
- Light Intensity (lux): Aprrox. 4400 to 6000 lux. measured at exposur initiation using a VWR Traceable light meter.
- Bulb type: Premira VitaLux fluorescent bulbs.
- Agitation: Continuous at a rate of 100 rpm +/- 10 rpm on an orbital shaker, rate monitored and recorded daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cells count were conducted on each replicate solution of the treatment levels and the controls using a Beckman Coulter Multisizer 4e Particle Analyzer.
- Observation of the health of the algal cells were made and recorded at each 24-hour interval. These observations were made in an alternating replicate of each control or treatment group using a hemacytometer (Neubauer Improved) and coumpound microscope.

- Spacing factor for test concentrations: 2.5
- Range finding study:
* Test concentrations: 0.0010 / 0.010 and 0.10 % of a 100 mg/L WAF.
* Results used to determine the conditions for the definitive study:
0.0010 % of a 100 mg/L WAF: percent inhibition compared to the control: 7%
0.010 % of a 100 mg/L WAF: percent inhibition compared to the control: 10%
0.10 % of a 100 mg/L WAF: percent inhibition compared to the control: 83%

Based on these results, nominal concentrations of 0.0020, 0.0051, 0.013, 0.032, 0.080, and 0.20% of a 100 mg/L WAF, and a control were selected for the definitive exposure.
Reference substance (positive control):
Zinc chloride (ZnCl2)

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
0.46 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L.: 1.8 - 2.3 µg/L
72 h
Dose descriptor:
Effect conc.:
> 0.032 - < 0.08 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
0.013 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Results based on measured concentrations were as follows:
72-hour average Specific Growth rate :
NOEC = 0.46 µg/L
LOEC = 1.4 µg/L
EC10 (95% CL)= 0.56 µg/L (0.38 - 0.73)
EC20 (95% CL) = 0.94 (0.73 - 1.1)
EC50 (95% CL) = 2.0 (1.8 - 2.3)

72-hour Yield:
NOEC = 0.46 µg/L
LOEC = 1.4 µg/L
EC10 (95% CLa)= NRb
EC20 (95% CL) = NR
EC50 (95% CL) = 1.1 µg/L (0.66 - 1.2)

CL= Confidence Limits
NR= Not Reported; lower 95% confidence limit could not be determined.

Test solutions without algae are used to examine the stability of the substance during the test period. Measured concentrations of the test solutions without algae after 72-h were only 50% of the nominal concentrations. In the presence of the algae clearly even lower concentrations were measured (decline by > 80%). The substance is not rapidly biodegradable, nor does it hydrolyse, indicating that the observed decrease was probably due to adsorption to glass vessels and/or algae or metabolisation by the algal biomass. Amounts of the test substance sorbed to the algae and/or to the walls of the glass containers might have been the cause for the large decrease in test concentrations during the test duration of the study and was considered in the interpretation of the results. Therefore, the results are also expressed in terms of nominal concentrations.

Results with reference substance (positive control):
The results was within the expected range for Raphidocelis subcapitata; therefore, the culture is considered of appopriate health and sensitivity for use in toxicity testing.
- Reference Substance: Zinc chloride (ZnCl2)
- Duration: 72 hours
- Date Performed: 10 to 13 July 2018
- EC50: 0.083 mg Zn/L, with a 95% confidence interval of 0.078 to 0.088 mg Zn/L (historical mean = 0.088 mg Zn/L, March 2005 to present)
Reported statistics and error estimates:
Prior to NOEC and LOEC determinations, the data were first checked for normality using Shapiro-Wilk’s Test and for homogeneity of variance using Bartlett's Test. If the data sets passed the tests for homogeneity and normality, then a parametric statistical test, e.g., Dunnett’s Multiple Comparison Test was used to determine the NOEC and LOEC. If the data did not pass the tests for homogeneity and normality, then the NOEC and LOEC were determined using an appropriate non-parametric statistical test, e.g., Dunn’s Test with Bonferroni-Holm’s Adjustment. All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilk’s and Bartlett's Tests, where the 99% level of certainty was applied.

If possible, EC10, EC20, EC50 values were calculated using a nonlinear regression model. The test concentrations bracketed the EC values so that the EC value came from interpolation rather than an extrapolation. The EC10, EC20,EC50 values were estimated so that (i) the 95% confidence limits reported for the EC value did not contain zero and was not overly wide, (ii) the 95% confidence interval for the predicted mean at the EC10, EC20, or EC50 value did not contain the control mean and (iii) there was no significant lack-of-fit of regression model to the data. If no concentration resulted in a 10, 20, or 50% reduction, the EC values were empirically estimated to be greater than the highest concentration tested. CETIS Version 1.8 (Ives, 2013) was used to perform all statistical calculations. These analyses followed the procedures described in the document, Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application (OECD, 2006) and the OECD Guideline for Testing of Chemicals No. 201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test (OECD, 2011).

Any other information on results incl. tables


Nominal Concentration

(% of a 100 mg/L WAF)

Mean 72-Hour Cell Density


Percent Inhibitiona













a     Percent inhibition compared to the control.

b     Cells were observed to be normal.

c     Cells were observed to be bloated.

d     NA = Not Applicable

Based on these results, nominal concentrations of 0.0020, 0.0051, 0.013, 0.032, 0.080, and 0.20% of a 100 mg/L WAF, and a control were selected for the definitive exposure.

Definitive Exposure

72-Hour Exposure of Raphidocelis subcapitatato ErC10, ErC20, ErC50, No-Observed-Effect Concentration (NOEC), and Lowest-Observed-Effect Concentration (LOEC) Values

Results Based on Geometric Mean Measured Concentrations (µg/L)

72-Hour ErC Value

95% Confidence Limits















72-Hour NOEC =


72-Hour LOEC =



Acceptance Criteria for the Control

Study Results

Criterion Met (Yes/No)

At exposure termination (72 hours), the cell density in the control should increase by a factor of 16 times.

Control cell density at 72 hours = 690,108 cells/mL (corresponding to a 69-times increase)


The mean coefficient of variation (CV) for section‑by‑section specific growth rates (day 0 to 1, 1 to 2, and 2 to 3) in the control replicates should not exceed 35%. This criterion applies to the mean value of the CV calculated for each control replicate.



The CV for the average specific growth rate of the control for the entire test period (0 to 72 hour growth rate) should not exceed 7%.



Applicant's summary and conclusion

Validity criteria fulfilled:
The objective of this study was to determine the effect of the test item on the growth of the freshwater green alga, Raphidocelis subcapitata under static conditions. The testing was performed according to the OECD Guideline for Testing of Chemicals. Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Guideline 201.
During the test a decrease of the concentration is observed of > 80%. The decrease observed could be assigned to sorption or other processes. As this may also occur under environmental conditions the nominal concentration is considered as the concentration to be used for the dose effect assesment. It is considered realistic to relate the observed effects to the nominal values. The 72-h ErC50 based on nominal values was between 32 and 80 µg/L (0032-0.08 mg/L). The 72-h NOErC based on nominal values is 13 µg/L (0.013 mg/L).