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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: ATCC-22662
- Source (laboratory, culture collection): National Institute Environmental Research, Korea
- Age of inoculum (at test initiation): 7 days
- Method of cultivation: The flasks then were placed on a shaker incubator at 170 rpm and 25°C, with 24 h light supplied via worm white fluorescent tubes, with an average illumination of 30 ± 5µE/m2s.


ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Stock alga was cultivated in 250 mL Erlenmeyer flasks, containing 200 mL sterilized nitrate-enriched BBM medium prepared in triple distilled water, to avoid nitrogen limitation during the high-density culture. The culture flask was agitated on a shaker at 170 rpm, and bubbled with air (1 vvm), without sparger. Light was continuously supplied, with an average of 30 ±5 µE/m2s, using warm-white fluorescent tubes located on top of the shaker. All the flasks were maintained in the shaker incubator at 25 ± 5°C for 7 days.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
25°C
Nominal and measured concentrations:
no details
Details on test conditions:
TEST SYSTEM

Experiments were performed in 250 mL Erlenmeyer flasks containing 55 mL of sterilized culture medium, inoculated with 5 mL samples of 7 day cultured algae. Solutions of organic solvents were subsequently added to the test flasks. The organic solvents tested were completely miscible with water, forming a homogeneous phase. Concentrations were adjusted in the range between 0.1 M and 1.26 µM to obtain complete concentration-response curves. At each determined exposure date, the optical density of the algal biomass was estimated at 438 nm using a spectrophotometer. The dry cell weight, corresponding to the optical density, was determined from the linear relationship; dry cell weight (g/L) = 0.329 x optical density. All experiments were performed in duplicate, but the control experiments were conducted in triplicate.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
ca. 22 000 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth rate
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:
other: Guideline study; GLP 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.

Description of key information

TMBX is hydrolytically unstable and breaks down to form methanol and boric acid in the presence of water, these species can be expected to be found folllowing release to the environment. Therefore, an assessment of ecotoxicological potential was conducted taking account of the effects of the hydrolysis breakdown products of TMBX upon the different trophic levels.

The 96 hour toxicity of methanol to Pseudokirchneriella subcapitata was established in a static system (Cho et al 2008), the ErC50 was determined to be 18260 mg/L (nominal) based on growth rate.

The acute aquatic toxicity of borates to freshwater algae (Pseudokirchneriella subcapitata) was examined under static conditions by Hanstveit and Oldersma (2000). The calculated ErC50 at 72hrs was 52.4 mg/L expressed as elemental Boron. The respective EC10 and NOEC were 35 mg/L and 17.5 mg/L.

The acute aquatic toxicity to marine algae (Phaeodactylum tricornutum) was determined in a static system (Rebstock et al 2011). The 72-hour ErC50, based on mean measured total boron concentrations, was estimated to be 66 mg B/L with 95% confidence limits of 63.5 and 67.7 mg B/L; The 72-hour EC10 and NOEC values were 50.7 and 27.9 mg B/L, respectively, based on total measured boron concentrations.

Based on the above results, the ErC50 and NOEC values for boron were used to calculate equivalent values for TMBX:

Freshwater 72 hr ErC50= 52.4 x (173.5/32.4) = 280.6 mg/L

Freshwater 72 hr NOEC=17.5 x (173.5/32.4) = 93.7 mg/L

Seawater 96 hr ErC50= 66 x (173.5/32.4) = 353.4 mg/L

Seawater 72 hr NOEC=27.9 x (173.5/32.4) = 149.4 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
280.6 mg/L
EC50 for marine water algae:
353.4 mg/L
EC10 or NOEC for freshwater algae:
93.7 mg/L
EC10 or NOEC for marine water algae:
149.4 mg/L

Additional information