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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:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: US EPA 850.5400, 1996 draft
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken at 0 and 96 h. Due to the high LOQ of 700 µg KMPS triple salt/L, samples from the highest concentrations and from stock solutions were taken only.
Vehicle:
no
Details on test solutions:
not indicated
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Species/strain: Marine diatom (Skeletonema costatum )
- Source: Department of Botany, University of Texas
- Method of cultivation: Cultures were prepared from stock culture and maintained at 20°C at 14 hours light
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
not indicated
Hardness:
not indicated
Test temperature:
20±2°C
pH:
8.1
Dissolved oxygen:
Not reported
Salinity:
20 ± 3 ‰
Nominal and measured concentrations:
Initial concentrations of test substance: 0, 55.6, 111, 222, 444, 889, 1780 µg KMPS/L, corresponding to
0, 25, 50, 100, 200, 400, 800 µg KHSO5/L

Measured concentrations:
The highest concentration of the solutions was analysed:
0 h and 96 h: below LOQ (LOQ = 700 µg/L)

Concentrations of stock solution ranged from 88 to 100% of nominal.

More details are given in "Any other information on materials and methods incl. tables"



Details on test conditions:
TEST SYSTEM
- Volume of test vessels: 250 mL vessels containing 100 mL test solution
- Vessels were covered with foam stoppers
- Aeration: Test chambers were hand-swirled twice daily
- Initial cells density: 7.6 x 10^4 cells/mL
- Control end cells density: 155 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, Saltwater algae nutrient medium (SWAM)

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, pH adjusted to 8.1 using 0.1 N hydrochloric acid and 0.1 N sodium hydroxide
- Photoperiod: 14 h light
- Light intensity: 4105 - 4351 lux
- Salinity (for marine algae): 20 ± 3 ‰

EFFECT PARAMETERS MEASURED : cell number, inhibition of growth rate
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
111 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
444 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
other: ErC50
Effect conc.:
556 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 511 - 600
Duration:
96 h
Dose descriptor:
other: ErC50
Effect conc.:
489 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 489 - 511
Details on results:
- Exponential growth in the control: No abnormal appearance of control algae was observed. The growth rate corresponded to the required range.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Statistics was performed with SAS software, the NOEC were determined using ANOVA and one-tailed Dunnett´s test.

Cell concentration data






































































































Test-Substance Concentration
(nominal)


 [µg KMPS triple salt/L]



Cell concentrations (mean values)
[cells /mL x 104]



measured



24 h



48 h



72 h



96 h



% inhibition


72 h



% inhibition


96 h



Control



25



54



127



155



-



-



55.6



22



55



117



150



5



6



111



23



48



112



174



12



5



222



15*



43



114



130



21



18



444



13*



31*



113



126



33



24



889



4.2*



4.8*



7.7*



7.2*



100



100



1780



3.8*



3.8*



8.7*



7.4*



100



100



Temperature [0C]



21.4-21.7



-



-



-



-



20.7-21.0



pH



8.1



-



-



-



-



7.9-8.8



An initial cell concentration of 7.6 x 104 wasincubated


* Statistically significant different from control (Dunnett´s test, p=0.05)


 


 


Growth rate data




















































































Test-Substance Concentration
(nominal)


 [µg KMPS triple salt/L]



Mean growth rate
[cells /mL/h]



measured



24 h



48 h



72 h



96 h



% inhibition


72 h



% inhibition


96 h



Control



0.048



0.040



0.039



0.031



-



-



55.6



0.044



0.041



0.038



0.031



3



0



111



0.047



0.038



0.037



0.032



5



-3



222



0.029*



0.036



0.038



0.029



3



6



444



0.023*



0.028*



0.038



0.029



3



6



889



0*



0*



0*



0*



100



100



1780



0*



0*



0.0018*



0*



95



100



 


 


* Statistically significant different from control (Dunnett´s test, p=0.05)


Area under the growth curve




















































































Test-Substance Concentration
(nominal)


 [µg KMPS triple salt/L]



Mean area under the growth curve



measured



24 h



48 h



72 h



96 h



% inhibition


72 h



% inhibition


96 h



Control



205



960



2940



6140



-



-



55.6



169



902



2780



5790



5



6



111



189



862



2600



5850



12



5



222



92.8*



610*



2320*



5060*



21



18



444



68.8*



422*



1980*



4660*



33



24



889



0*



0*



0*



0*



100



100



1780



0*



0*



0*



0*



100



100



* Statistically significant different from control (Dunnett´s test, p=0.05)


 


 


Effect data as KMPS triple salt
































-



96 h [µg/L]1



95 % c.l.



EbC50



489



489-511



ErC50



556



511-600



NOEC biomass



111



-



NOEC growth



444



-




1 effect data are based on nominal concentrations

Validity criteria fulfilled:
yes
Conclusions:
The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).The ErC50 and EbC50 values were calculated to be 556 and 489 µg KMPS triple salt/L, respectively.
Executive summary:

Materials and methods


The acute toxicity of KMPS triple salt to the marine diatom was determined in a static dose-response 96-h test according to the US EPA guideline 850.5400, 1996.


 


Results and discussion


The analytical verification of stock solutions resulted in recoveries ranging from 88 100 % of nominal. The measured concentrations in the 1,780 µg KMPS triple salt/L treatment (corresponding to 800 µg KHSO5) at 0 and 96 hours were less than the LOQ, which was determined to be 700 µg KHSO5/L/. The results were based on nominal concencentrations.


NOEC  = 111 µg KMPS triple salt/L (biomass), 444 µg KMPS triple salt/L (growth rate)


ErC50 = 556 µg KMPS triple salt/L


EbC50 = 489 µg KMPS triple salt/L           

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-05-04 - 2001-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Triplicate samples were taken at 0 hours from stock solutions. Samples were not filtered, i.e. to remove algal cells before analysis.
Vehicle:
no
Details on test solutions:
not indicated
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Species/strain: Selenastrum capricornutum, Strain No. CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (6000-10000 lux) at 23°C
Test type:
static
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
not indicated
Hardness:
not indicated
Test temperature:
23±1°C
pH:
6.9 – 7.1
Dissolved oxygen:
8.7 – 9.5
Salinity:
not indicated
Nominal and measured concentrations:
Nominal test solutions: 0.0625, 0.125, 0.25, 0.50, 1.0 mg/L

Nominal concentrations of stock solutions, which were analysed: 1.25, 2.5, 5.0, 10 mg/L
Measured concentrations of stock solutions: 1.3, 2.8, 5.3, 10 mg/L
More details are given in "Any other information on materials and methodes incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessels were loosely stoppered
- Size of the test vessels: 250 mL
- Aeration: by orbital shaker, oscillating at 140 cycles per minute
- Initial cells density: 10.000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to the guideline
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily
- Light intensity and quality: 3600-4700 lux

EFFECT PARAMETERS MEASURED: growth inhibition and biomass
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
0.97 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.87 - > 1.0
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.0 - > 1.0
Duration:
96 h
Dose descriptor:
other: EbC50
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.92 - > 1.0
Duration:
96 h
Dose descriptor:
other: ErC50
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Not determined
Details on results:
- Exponential growth in the control: yes
- Other effects: Regrowth was observed after 3 days when cultures previously exposed to 1.0 mg/L were placed in fresh medium. This indicates that
KMPS triple salt compound had an algistatic effect on algal growth at a concentration of 1.0 mg/L.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
EC50 values calculated using logistic model (Ashton, 1972), 95% confidence limits estimated by likelihood ratio method (Williams, 1986)

Cell concentration data




















































































Test-Substance Concentration
(nominal) [mg/L]



Cell concentrations (mean values)
[cells/mL]



measured



0 h



24 h



48 h



72 h



96 h



Control



16495



39881



134907



536167



1682000



0.0625



15460



37316



139713



569867



1661400



0.125



14629



36056



139040



571853



1900267



0.25



13459



34380



112883



499667



1676900



0.50



12630



34397



119847



541707



1728200



1.0



12046



29361



67633



229900



879000



Temperature [0C]



23



23



23



23



22



pH



7.5-7.6



-



-



-



7.4-7.6



 


 


Summary of effects on cell growth and biomass after 72 h























































Nominal concentration (mg/L)



Area under curve 72 h



% inhibition



Growth rate


0-72 h



% inhibition



Control



9.6



-



0.048



-



0.0625



10



-5.4



0.050



(3.7)



0.125



10



-5.4



0.051



(5.4)



0.25



8.7



9.5



0.050



(3.8)



0.50



9.4



2.0



0.052



(8.1)



1.0



4.4



55



0.041



15



Figures in brackets denote negative inhibition, which indicate increase  in cell density compared to control


 


 


Summary of effects on cell growth and biomass after 96 h























































Nominal concentration (mg/L)



Area under curve 72 h



% inhibition



Growth rate


0-72 h



% inhibition



Control



36



-



0.048



-



0.0625



37



-2.0



0.049



(1.2)



0.125



40



-10



0.051



(5.2)



0.25



35



3.7



0.050



(4.3)



0.50



36



-1.4



0.051



(6.4)



1.0



17



52



0.045



7.2



Figures in brackets denote negative inhibition, which indicate increase  in cell density compared to control


 


 


Effect data


































-



72 h [mg/L]1



95 % c.l.



96 h [mg/L]1



 95 % c.l.



EbC50



0.97



0.87->1.0



1.0



0.92->1.0



ErC50



>1.0



0.0->1.0



>1.0



n.d.



NOEC



0.50



-



0.50



-



1effect data are based on nominal concentrations

Validity criteria fulfilled:
yes
Remarks:
(Factor of increase of cell numbers > 16; pH of control cultures remained within 1 unit; Concentration of test substance equal with or greater than 80% of initial concentration during test)
Conclusions:
The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). The 72-h EC50 for algae was determined to be 0.97 (biomass) mg/L and >1.0 (growth rate). All results are expressed in terms of nominal concentration.
Executive summary:

Materials and methods


An acute toxicity test in algae was performed according to OECD 201 without deviations. The test system was static and Selenastrum capricornutum was used as test organism. Algae were exposed for 72 h to 5 different concentrations in order to derive an EC50.


 


Results


NOEC (72 hours) = 0.5 mg/L


EbC50 (72 hours) = 0.97 mg/L


ErC50(0-72 hours) > 1.0 mg/L


EbC50(96 hours) = 1.0 mg/L


ErC50 (0-96 hours) > 1.0 mg/L

Description of key information

The acute toxicity of KMPS triple salt to aquatic algae was tested in two key studies. One acute toxicity test was performed according to the EU Guideline C3 and to the OECD Guideline 201. The second test was performed according to the US EPA guideline 850.5400, 1996.

Key value for chemical safety assessment

EC50 for freshwater algae:
1 mg/L
EC50 for marine water algae:
0.556 mg/L
EC10 or NOEC for freshwater algae:
0.5 mg/L
EC10 or NOEC for marine water algae:
0.444 mg/L

Additional information

The acute toxicity tests in algae and diatoms were performed according to guideline OECD 201, without deviations.The test systems were static and Selenastrum capricornutum (freshwater) and Skeletonema costatum (marine) were used as test organisms.Algae and diatoms were exposed for 72 h to 5 different concentrations in order to derive EC50-values.


It is important to note that the marine test is likely to overestimate the toxicity to KMPS triple salt, because in salt water oxidation of KMPS triple salt generates chlorine from sodium chloride, increasing the biocidal activity of test solutions. Further, as results obtained from these exponential test systems cannot be used without logarithmic transformation, only growth rate ErC50-values are relevant (TGD, 2003). Based on the behaviour of KMPS triple salt in sea water and the marginal ErC50-values obtained, classification and labelling with Acute env. tox. cat. 1, H400, according to Regulation 1272/2008/EC (CLP), respectively, does not apply.