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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 to 16 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Qualifier:
according to guideline
Guideline:
other: ASTM E1218-97a Standard Guide for Conducting Static 96-h Toxicity Tests with Microalgae
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were made on samples collected at 0 h and 72 h. Samples at 0 h were taken from parent solutions. Samples at 72 h were taken from combined replicate samples. To remove algae, 72 h samples were centrifuged and supernatant removed for analysis
Vehicle:
no
Details on test solutions:
Test medium was synthetic seawater (Marinemix, Wiegandt GmbH) prepared by addition to autoclaved lab reagent water, with a saltwater algal nutrient medium enrichment. Boron concentrations in this medium (geomean 7.7 mg B/L) exceeded the boron levels typically expected in natural seawater (ca 4.5 mg B/L).
Test organisms (species):
Phaeodactylum tricornutum
Details on test organisms:
Stock obtained from University of Texas at Austin (UTEX). Cultures maintained in 14 h light: 10 hr dark at 20 degrees C. Four days prior to initiation of definitive test, new culture was cloned and incubated under continuous illumination of approximately 8000 lux.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
20.5 to 21.0 degrees C
pH:
7.2 to 8.1
Salinity:
30 +/- 2 ppt
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 6.3, 13, 25, 50 and 100 mg B/L (as added boron)
Geometric Mean Measured Total Concentrations: 7.68 (control), 14.7, 23.3, 35.6, 70.1, and 109 mg B/L
Geometric Mean Calculated Added Concentrations: 0 (control), 7.0, 15.6, 27.9, 62.4, 101.3 mg B/L
Details on test conditions:
The exposure flasks were 250-mL Erlenmeyer flasks with foam stoppers. Prior to test initiation, the flasks were cleaned and autoclaved according to ABC standard operating procedures. The control was replicated six times and each test substance treatment was replicated three times. Each replicate contained 100 mL of the appropriate parent solution. An additional replicate (replicate D) of the 6.3 mg B/L test substance treatment, containing 100 mL of the appropriate parent solution, was also prepared and used to evaluate the potential for incorporation of the test substance into the algal biomass. At test initiation, all replicates of the controls and each A, B, and C replicate of each test substance treatment were inoculated with 1.0 mL of an algal concentrate containing approximately 1.0 x 10^6 cells/mL, resulting in a final density of approximately 1.0 x 10^4 cells/mL for each flask. The replicates were inoculated with algae within 30 minutes after test solution preparation. At 24, 48, and 72 hours (±1 hour), cell density was measured by direct microscopic counting with a hemacytometer. Replicate D of the 6.3 mg B/L test substance treatment was not inoculated with algae.
During the three-day exposure period, the flasks were randomly positioned daily using a computer-generated random number table and incubated at 20 ± 2°C in a temperature controlled environmental chamber under continuous cool-white fluorescent lighting. A continuous recording of environmental chamber temperature was made from one uninoculated blank flask using an electronic datalogger with thermistor probe. Light intensity was measured daily with a LI-COR Model LI-189 light meter equipped with a LI-COR photometric sensor and ranged from 8,014 to 8,254 lux. The flasks were swirled on an orbital shaker table at 100 rpm throughout the test. Temperature and pH were measured in all parent solutions prior to distribution of the solutions to the test flasks. At 72 hours, temperature and pH were measured in replicate A of the controls and each test substance treatment. All temperature and pH measurements of the test solutions were performed with a WTW Model pH 330i meter.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
50.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: added boron (46.0 to 55.4)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
66 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: added boron (63.5 to 67.7)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
27.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: added boron
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
41.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: added boron (40.9 to 42.8)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
54 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: added boron (52.9 to 55.2)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
27.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: added boron
Duration:
62.4 h
Dose descriptor:
LOEC
Effect conc.:
70.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: growth rate and biomass
Remarks on result:
other: added boron
Details on results:
Control algal growth met acceptable limits. Cell numbers were greater than 16 times the initial density. Coefficient of variation among control replicates did not exceed 7% and pH did not increase more than 1 unit. All water quality parameters remained within acceptable limits.
Reported statistics and error estimates:
Statistical analysis based on one-way ANOVA followed by Dunnett's test. ECx estimates obtained from probit method and trimmed Spearman-Karber method if poor fit in the probit approach.

Algal Endpoint values

Group Nominal Concentration (mg B/L) Geomean measured (mg B/L) Cell Density 72-h (cells/mL x 10e4) Mean Growth Rate (cells/mL-h) Yield (cells/mL x 10e4)
0 7.7 42 0.052 40.8
T1  6.3 14.7 45 0.053 43.7
T2  13 23.3 43 0.052 41.7
T3  25 35.6 41 0.052 39.7
T4  50 70.1 9.9* 0.032* 8.9*
T5  100 109.0 1.1* 0.001* 0.1*
* Significant difference from control (p<0.05)
Validity criteria fulfilled:
yes
Conclusions:
Growth inhibition of the marine diatom Phaeodactylum tricornutum exposed to boric acid was measured. Cell densities were measured daily and used to determine growth rate and biomass yield. Based on total boron measured values, NOECs were 35.6 mg B/L and lower, EC10-growth was 58.4 mg B/L, EC50-growth was 73.3 mg B/L, EC10-yield was 49.5 mg B/L, and EC50-yield was 61.7 mg B/L. Based on added boron measured values, NOECs were 27.9 mg B/L and lower, EC10-growth was 50.7 mg B/L, EC50-growth was 65.6 mg B/L, EC10-yield was 41.8 mg B/L, and EC50-yield was 54.0 mg B/L. The test met acceptability criteria for the method, based upon ISO 10253:2006.
Executive summary:

Growth inhibition of the marine diatom Phaeodactylum tricornutum exposed to boric acid was measured. Cell densities were measured daily and used to determine growth rate and biomass yield. Based on total boron measured values, NOECs were 35.6 mg B/L and lower, EC10-growth was 58.4 mg B/L, EC50-growth was  73.3 mg B/L, EC10-yield was 49.5 mg B/L, and EC50-yield was 61.7 mg B/L. Based on added boron measured values, NOECs were 27.9 mg B/L and lower,  EC10-growth was 50.7 mg B/L, EC50-growth was 65.6 mg B/L, EC10-yield was 41.8 mg B/L, and EC50-yield was 54.0 mg B/L. The test met acceptability criteria for the method, based upon ISO 10253:2006 and was conducted according to GLP requirements.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At the start and end of the growth inhibition test, single 50 ml samples were taken from the control series without algae containing nominal test substance concentrations of 0, 32, 100 and 320 mg/L. The samples were placed in a refrigerator until transfer to the analytical sciences Division for analyses.

One sample was taken from each flask after 0, 26.0, 49.5 and 74.5h and the number of algal cells per ml in the samples was analysed.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source: ATCC 22662, supplied by the 'American Type Culture Collection', 12301 Parklawn Drive, Rockville, Maryland 20852, USA.
- A preculture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
74.5 h
Hardness:
24.2 mg CaCO3/L
Test temperature:
23 +- 2°C
pH:
at test initiation: 7.5-8.3
at end of test: 8.0-8.3
Nominal and measured concentrations:
Nominal concentrations : 0, 32, 56, 100, 181, 321, 562 mg/L boric acid => 0, 5.6, 9.8, 17.5, 31.7, 56.2, 98.4 mg B/L
The measured concentrations were found to be 95.4 -94.7 % of the nominal concentrations, and they show a linear relationship with the nominal concentrations. The test substance also appeared to be stable during the test, and therefore nominal concentrations were used to report the test results according to the EU C.3 Guideline.
The samples were analysed for B using inductively coupled atomic emission spectrometry (ICP-AES) at a wavelength of 249.704 nm. The limit of detection is 0.03 mg/L, the limit of determination 0.10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 200 ml conical test flasks covered with silicone sponge caps
- Initial cells density: 10^6 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: OECD guideline medium (1984) with 150 mg/L NaHCO3 instead of 50 mg/L and addition of Fe-citrate


OTHER TEST CONDITIONS
- Light intensity and quality: fluorescent lamps within standard of 60-120 µmol/(s.m²)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer IIe


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.10, 1.0, 10, 32.8, 102, 328 and 1025 mg/L
Duration:
3 d
Dose descriptor:
EC10
Effect conc.:
24.5 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: Effect concentration with regard to the area under the growth curves; analytical confirmation of B concentrations; confidence limits: 17.4 - 31.4 mg/L
Duration:
3 d
Dose descriptor:
EC10
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: Effect concentration with regard to the growth rate during exponential growth; analytical confirmation of B concentrations
Duration:
3 d
Dose descriptor:
NOEC
Effect conc.:
17.5 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: analytical confirmation of B concentrations
Duration:
3 d
Dose descriptor:
EC50
Effect conc.:
40.2 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: Effect concentration with regard to the area under the growth curves; analytical confirmation of B concentrations; confidence limits: 31.5-55.9 mg/L
Duration:
3 d
Dose descriptor:
EC50
Effect conc.:
52.4 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth rate
Remarks on result:
other: Effect concentration with regard to the growth rate during exponential growth; analytical confirmation of B concentrations; confidence limits: 45.4 - 59.4 mg/L
Details on results:
NOEC and EC10 values are provided for the endpoints reported, i.e. biomass and growth rate. In general preference is given to the use of EC10 values for PNEC derivation. However, for growth rate no confidence limits could be calculated. therefore the NOEC for growth rate was selected. For algae, the endpoint growth rate is preferred over the endpoint biomass.
Reported statistics and error estimates:
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.
For the NOEC calculations were carried out using the DEBtox software package according to the Dynamic Energy Budgets Theory developed by Kooijman and Bedeaux. Model parameters for population growth and their asymptotic standard deviation and correlation coefficients were estimated. The NEC was calculated from the profile ln likelihood function.

The tests fulfilled the validity criteria of sufficient growth (control growth rate was 0.054 h-1) and a minimum increase of test medium pH (highest pH was 8.3)

Validity criteria fulfilled:
yes
Conclusions:
The EC10 for growth rate was 35 mg B/L and the EC50 growth rate was 52.4 mg B/L.
Executive summary:

The test item boric acid has been evaluated for its toxicity towards the fresh water alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) in an Alga growth inhibition test according to OECD 201 under GLP requirements. The nominal concentrations were 0, 32, 56, 100, 181, 321, 562 mg/L boric acid , according to 0, 5.6, 9.8, 17.5, 31.7, 56.2, 98.4 mg B/L. The exposure duration was 72 hours under static conditions. The EC10 for growth rate was 35 mg B/L, the NOEC growth rate was 17.5 mg B/L and the EC50 growth rate was 52.4 mg B/L.

Description of key information

The test item boric acid has been evaluated for its toxicity towards the fresh water alga Pseudokirchneriella subcapitata

(formerly Selenastrum capricornutum) in an Alga growth inhibition test according to OECD 201 under GLP requirements. The nominal concentrations were 0, 32, 56, 100, 181, 321, 562 mg/L boric acid , according to 0, 5.6, 9.8, 17.5, 31.7, 56.2, 98.4 mg B/L. The exposure duration was 72 hours under static conditions. The EC10 for growth rate was 35 mg B/L, the NOEC growth rate was 17.5 mg B/L and the EC50 growth rate was 52.4 mg B/L.

In another study the growth inhibition of the marine diatom Phaeodactylum tricornutum exposed to boric acid was measured. Cell densities were measured daily and used to determine growth rate and biomass yield. Based on total boron measured values, NOECs were 35.6 mg B/L and lower, EC10 -growth was 58.4 mg B/L, EC50 -growth was  73.3 mg B/L, EC10 -yield was 49.5 mg B/L, and EC50 -yield was 61.7 mg B/L. Based on added boron measured values, NOECs were 27.9 mg B/L and lower,  EC10 -growth was 50.7 mg B/L, EC50 -growth was 65.6 mg B/L, EC10 -yield was 41.8 mg B/L, and EC50 -yield was 54.0 mg B/L.

The test met acceptability criteria for the method, based upon ISO 10253:2006 and was conducted according to GLP requirements.

Please also refer to read-across statement in section 13.

Key value for chemical safety assessment

EC50 for freshwater algae:
52.4 mg/L
EC10 or NOEC for freshwater algae:
17.5 mg/L

Additional information