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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:
March 10, 2003 to November 3, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 1984
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
The sample of 3-chloro-o-xylene (T.R. Wilbury Laboratories sample number 1763) used for the study was delivered on March 10, 2003. It was contained in four 50 mL amber bottles that were shipped in a plastic bucket at ambient temperature. The Label attached to the bottles included the following information: "3-Cl-1,2-dimethylbenzene, CAS # 608-23-1, Clariant Lot 26.11.02, 25 g, 98%, combustible". 3-chloro-o-xylene (a colorless liquid) was supplied by General Electric Corp., 1 Research Circle, Niskayuna, New York. Test concentrations were corrected for the purity of the test substance. Prior to use the test substance was stored at room temperature in the dark. The stability of the test substance was determined under test conditions by the analysis of test solutions during the definitive toxicity test.
Analytical monitoring:
yes
Details on sampling:
Analytical determination of test substance concentration (active ingredient) was performed with samples collected from stock solutions prepared at each concentration prior to their distribution to test vessels at the start of the toxicity test and from each replicate test vessel after 72 hours. Samples collected from replicate test vessels at the end of the test were pooled. All samples collected at the end of the test were centrifuged at the time of collection to remove algal cells. Each sample set was accompanied by three matrix spike samples prepared in dilution water, a dilution water blank, and the stability blank.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
A stock solution with a nominal concentration of 100 000 mg/L was prepared by bringing 5.0828 g of test substance to a total volume of 50 mL with dimethylformamide in a glass volumetric flask. This stock solution was used to, prepare 70 000, 35 000, 18 000, 9 100, and 4 600 mg/L dimethylformamide stock soutions in 10 mL Class A volumetric flasks, and those stock solutions were then used to prepare 7.0, 3.5, 1.8, 0.91 and 0.46 mg/L test solutions by transferring 0.050 mL of stock solution into 500 mL of algal media. The pH of the solutions was adjusted to 7.5 ± 0.1 using 1N sodium hydroxide and 0.1N hydrochloric acid. A stability blank sample set up at 7.0 mg/L was placed among the test vessels. No algae was inoculated into the stability blank. The dimethylformamide concentration of solvent control was 0.1 mL/L dimethylformamide.
Test organisms (species):
Selenastrum sp.
Details on test organisms:
Algae used for the test (Selenastrum capricornutum, UTEX 1648) were from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin and delivered to T.R. Wilbury Laboratories on July 17, 2001. The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. During the 14 day acclimation period prior to the start of the test, the temperature ranged from 23.7 to 24.4°C and the light intensity ranged from 50 to 51 µEin/m2sec (24 hours light/0 hours dark photoperiod).
During the acclimation period, the culture was actively growing in at least two subcultures prior to the start of the toxicity test. The subsample of algae used to inoculate media at the start of the definitive test came from a four day old culture. Identification of the culture organisms, which are also referred to as Raphidocelis subcapitata, was verified using an appropriate taxonomic key.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
96 to 144 hours
Test temperature:
24.0 to 24.3°C
pH:
7.5 ± 0.1.
Nominal and measured concentrations:
Nominal: 0.46, 0.91, 1.8, 3.5 and 7.0 mg/L
Measured (geometric mean): 0.277, 0.661, 1.21, 2.40 and 4.87 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL clear glass vials
- Type (delete if not applicable): closed
- Headspace, fill volume: vials were capped to eliminate all headspace; 40 mL
- Aeration: no
- Initial cells density: 10 000 cells/mL
- Control end cells density: 427 000 cells/mL
- No. of organisms per vessel: 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, sterile freshwater AAP medium

TEST MEDIUM / WATER PARAMETERS
- Test medium/dilution water: Water used for acclimation of test organisms and for all toxicity testing was sterile freshwater AAP medium (U.S. EPA, 1978) at a pH of 7.5 ± 0.1 (the pH was adjusted with 0.1N HCl and 0.1N NaOH). Dilution water collected at the start of the definitive toxicity test had a measured total organic carbon concentration of 1.2 ppm. Intervals of analytical water quality measurement are 2 times a year.
- Water parameters: Temperature of the incubator was measured and recorded daily (thermometer number 2968) and the temperature in a representative vessel of water incubated with the test vessels was continuously recorded. The pH of test solutions was measured (Beckman meter number 209 pHT-19) , and recorded in each test solution prior to distribution to the test vessels at the start of the test and in each test vessel used to enumerate algal cells at the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, prior test start
- Photoperiod: 24 hour light and 0 hour dark photoperiod
- Light intensity and quality: cool-white fluorescent lights, approximately 410 footcandles (also measured as approximately 54 to 55 µEin/m2sec)


TOXICITY TEST / EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a hemocytometer at 24, 48, and 72 hours. At the end of the toxicity test, a determination of whether the toxic effects observed at 3.46 and 7.07 mg/L were algistatic or algicidal was made by transferring 0.50 mL of test solution from each flask to 100 mL of dilution water without the test substance and incubating these flasks under test conditions for 96 to 144 hours. At the conclusion of this incubation period, the number of cells/mL was determined in each flask.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10, 100 and 1 000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 2.98 - 3.49
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CI: 1.28 - 1.63
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.66 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
biomass
Details on results:
The algal population grew well, resulting in an average of 427 000 cells/mL in the control alter 72 hours. No effects (relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted during the test. Water quality throughout the test was within acceptable limits. Exposure of Selenastrum capricornutum to 3-chloro-o-xylene for 72 hours resulted in the following EC50 values (and 95% confidence intervals) based on geometric mean measured concentrations: EbC50 (biomass) = 1.44 mg/L (95% CI: 1.28 - 1.63); ErC50 (growth rate) = 3.21 mg/L (95% CI: 2.98 - 3.49).

The 72 hour NOEC was 0.66 mg/L 3-chloro-o-xylene when determined using the number of cells/mL or the average specific growth rate. At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each 2.40 and 4.87 mg/L concentration was combined with 100 mL of fresh media and incubated under test conditions. During a 96 hour incubation period the algal population increased from approximately 1 880 cells/mL to 748 000 cells/mL at 2.40 mg/L and during a 144 hour incubation period the algal population increased from approximately 445 cells/mL to 422 000 cells/mL at 4.87 mg/L, indicating that the toxic effects were algistatic rather than algicidal.

The validity criteria of OECD 201 (adopted 2011) were fulfilled:
- According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. With the current test it was found to be 42.7. The test fulfilled this validity criterion.
- The mean of the replicate coefficients of variation in the section-by-section growth rate in the control was: 22.8% (solvent control: 31.4%). According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%. The test fulfilled this validity criterion.
- The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 2.4% (solvent control: 0.9%). According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%. The test fulfilled this validity criterion.

Insoluble material was not observed during the definitive toxicity test.

Note: Biological and analytical data generated by the acute toxicity test are given in any other information on results.

Results on analytical measurements
Nominal concentrations of 3-chloro-o-xylene were: 0 mg/L (control), 0.46, 0.91, 1.8, 3.5, and 7.0 mg/L. Initial measured concentrations were ND (control and solvent control), 0.410, 0.851, 1.78, 3.46, and 7.07 mg/L (Table 3), representing 89 to 101% of nominal concentrations. Final measured concentrations were ND (control), 0.187, 0.513, 0.827, 1.67, and 3.36 mg/L (41 to 56% recovery), indicating that the test substance was not stable under test conditions. Geometric mean measured concentrations were ND (none detected; control and solvent control), 0.277, 0.661, 1.21, 2.40, and 4.87 mg/L.
Reported statistics and error estimates:
A statistical re-evaluation of the raw data was performed using the software tool ToxRat Professional (v. 2.10) in 2017. A re-evaluation was performed because the five test concentrations used for the first assessment were based on initially measured concentration. However, according to the OECD 201 (2011) the following applies: “if the deviation from the nominal or measured initial concentration is not within the range of ± 20 %, analysis of the results should be based on geometric mean concentration during exposure or on models describing the decline of the concentration of the test substance.” Accordingly, in the statistical re-evaluation the calculated effect concentrations were based on geometric mean measured concentrations. ECx concentrations were calculated with Probit analysis using linear max. likelihood regression. The NOEC concentration were calculated with ANOVA using Williams Multiple Sequential t-test Procedure. Levels of significance were set to p = 0.5.

Cell growth data from the toxicity test with 3-chloro-o-xylene and the freshwater alga, Selenastrum capricornutum.

Measured concentration of test item (mg/L) *
Replicate Number of Cells per Milliliter
Hour of Exposure
0 24 48 72
Control 1 10000 48000 126000 426000
  2 10000 54000 134000 466000
  3 10000 41000 130000 390000
  mean 10000 48000 130000 427000
Solvent Control 1 10000 50000 132000 404000
  2 10000 52000 132000 382000
  3 10000 58000 152000 422000
  mean 10000 53000 139000 403000
0.28 1 10000 49000 124000 390000
  2 10000 52000 130000 370000
  3 10000 54000 138000 476000
  mean 10000 52000 131000 412000
  % control 100 108 101 96
0.66 1 10000 54000 106000 368000
  2 10000 39000 130000 358000
  3 10000 47000 148000 368000
  mean 10000 47000 128000 365000
  % control 100 98 98 85
1.21 1 10000 25000 64000 190000
  2 10000 32000 88000 262000
  3 10000 26000 66000 300000
  mean 10000 28000 73000 251000
  % control 100 58 56 59
2.40 1 10000 15000 70000 110000
  2 10000 22000 68000 122000
  3 10000 21000 56000 144000
  mean 10000 19000 65000 125000
  % control 100 40 50 29
4.87 1 10000 12000 26000 27000
  2 10000 <10000 23000 34000
  3 10000 <10000 19000 28000
  mean 10000 <11000 23000 30000
  % control 100 <23 18 7

* Geometric mean measured concentrations

Measured concentrations of 3-chloro-o-xylene during the toxicity test with the freshwater alga, Selenastrum capricornutum.

Nominal test item concentration (mg/L) Measured test item concentration (mg/L)
0 Hour  % Recovery  72 Hour  % Recovery Geometric mean 
Test Media Samples          
0.46 0.410 89 0.187 41 0.277
0.91 0.851 94 0.513 56 0.661
1.8 1.78 99 0.827 46 1.213
3.5 3.46 99 1.67 48 2.40
7 7.07 101 3.36 48 4.87
Stability Blank          
7 7.07 101 4.88 70 5.874
Matrix Spike Sample          
1.8 1.94 108 1.77 98 1.85
  1.98 110 1.59 88 98.39
  1.98 110 1.6 89 98.94
Blank          
0 ND ND ND - -
Validity criteria fulfilled:
yes
Remarks:
according to OECD 201, adopted 2011
Conclusions:
72h-ErC50 (Selenastrum capricornutum) = 3.21 mg/L; 72h-EbC50 (Selenastrum capricornutum) = 1.44; 72h-NOErC (Selenastrum capricornutum) = 0.66; 72h-NOEbC (Selenastrum capricornutum) = 0.66
Executive summary:

The purpose of this study was to determine the 72 hour median effective concentrations (EC50s) and the 72 hour no observed effect concentration (NOEC) of the test substance to algae. The test was performed according to OECD 201 and in compliance with GLP. The test was performed under static conditions with five concentrations of test substance and a dilution water control at 24 ± 2°C. The test was performed in sealed containers with no head space to minimize the loss of test substance to the atmosphere. Nominal concentrations of 3-chloro-o-xylene were 0 mg/L (control and solvent control), 0.46, 0.91, 1.8, 3.5, and 7.0 mg/L. Geometric mean measured test concentrations were 0.277, 0.661, 1.21, 2.40, and 4.87 mg/L. The initial measured concentrations were 89 to 101% of nominal concentrations, whereas final measured concentrations were 41 to 56% recovery, indicating that the test substance was not stable under test conditions. Therefore, effect concentrations were based on geometric mean measured concentrations (effect concentrations were re-calculated based on geometric mean measured concentration using raw data, because they were based on initially measured concentrations in the original study report).

Insoluble material was not observed at any tested concentration during the test. The dilution water was sterile freshwater AAP medium adjusted to a pH of 7.5 ± 0.1. The test was performed in clear glass 40 mL vials that contained 40 mL of test solution. Each day (except day 0), three replicates were randomly selected for the daily counts and then sacrificed. Exposure of algae to 3-chloro-o-xylene for 72 hours resulted in a median effective concentration based on biomass (EbC50) of 1.44 mg/L, and 3.21 mg/L based on the specific growth rate (ErC50). When treatment data were compared to the controls, the 72 hour NOEC was 0.66 mg/L 3-chloro-o-xylene for both endpoints, biomass and growth rate.

The validity criteria of OECD 201 (adopted 2011) were fulfilled, and the study was considered reliable and adequate for the environmental hazard assessment.

Description of key information

72h-ErC50 (Selenastrum capricornutum) = 3.21 mg/L; 72h-EbC50 (Selenastrum capricornutum) = 1.44; 72h-NOErC (Selenastrum capricornutum) = 0.66; 72h-NOEbC (Selenastrum capricornutum) = 0.66 (OECD 201, RL1)

Key value for chemical safety assessment

EC50 for freshwater algae:
3.21 mg/L
EC10 or NOEC for freshwater algae:
0.66 mg/L

Additional information

A reliable key study was performed according to OECD 201 and in compliance with GLP (RL1, 2003). The test was performed in sealed containers with no head space to minimize the loss of test substance to the atmosphere. Nominal concentrations of 3-chloro-o-xylene were 0 mg/L (control and solvent control), 0.46, 0.91, 1.8, 3.5, and 7.0 mg/L. Geometric mean measured test concentrations 0.277, 0.661, 1.21, 2.40, and 4.87 mg/L. Insoluble material was not observed at any tested concentration during the test. Exposure of algae to 3-chloro-o-xylene resulted in a 72h-EbC50 of 1.44 mg/L and ErC50 of 3.21 mg/L based on biomass and specific growth rate, respectively. The 72h-NOEC was 0.66 mg/L 3-chloro-o-xylene for both endpoints, biomass and growth rate. The validity criteria of OECD 201 (adopted 2011) were fulfilled, and the study was considered reliable and adequate for the environmental hazard assessment.