Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Collected from each replicate test vessel at the start and end of the test.
Vehicle:
yes
Details on test solutions:
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 NaOH). The vehicle used was dimethylformamide (DMF).

A series of solutions were prepared by bringing 0.0377, 0.0754, 0.1509, 0.3018 and 0.6038 g of test substance to 50 mL of dimethylformamide in volumetric flasks. The solutions were shaken vigorously until the test substance dissolved. Test solutions were prepared by combining 0.050 mL of each stock solution and dilution water in 500 mL volumetric flasks. A stability blank sample was set up at 1200 µg/L and placed among the test vessels. No algae were inoculated into the stability blank.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Algae used for the test: Selenastrum capricornutum, UTEX 1648.
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, the culture was actively growing in at least 2 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 seven 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:
96 h
Post exposure observation period:
none
Test temperature:
24.3 to 24.7 °C
pH:
7.2 to 7.3 (initial); 7.8 to 9.4 (termination)
Nominal and measured concentrations:
Nominal: 0 (control and solvent control), 75, 150, 300, 600 and 1200 µg/L
Mean Measured: ND (none detected; control and solvent control), 81, 161, 297, 595 and 1165 µg/L
Details on test conditions:
A range-finding test was conducted under static conditions with a control, a solvent control, and five concentrations of DMBPC: 0.0, 0.99, 9.9, 100, 500 and 1200 µg/L. At the conclusion of the test, the number of cells/mL in the test vessels equalled the following percent of the number of cells/mL in the control vessels: 0.99 g/L = 81 %; 9.9 µg /L = 79 %; 100 µg/L = 83 %; 500 µg/L = 64 %; and 1200 µg/L = 45 %.

The definitive toxicity test was conducted at 24 ± 2°C with five concentrations of test substance, a control, and a solvent control (0.10 mL/L dimethylformamide). A series of solutions were prepared by bringing 0.0377, 0.0754, 0.1509, 0.3018 and 0.6038 g of test substance to 50 mL of dimethylformamide in volumetric flasks. The solutions were shaken vigorously until the test substance dissolved.

These stock solutions were used to prepare the nominal test solutions by transferring the appropriate amount of stock solution into DMF. Water quality measurements were made, analytical samples were collected and each solution was subdivided into three clear glass 250 mL Erlenmeyer flasks. The flasks were inoculated with approximately 10 000 algal cells/mL and capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator during the test (a random numbers table was used to select the location for each vessel). A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 380 to 400 footcandles.

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer at 24, 48, 72, and 96 hours.

Temperature of the incubator was measured and recorded daily and the temperature in a representative vessel of water incubated with the test vessels was continuously recorded. The pH of test solutions was measured and recorded in each test solution prior to distribution to the test vessels at the start of the test and in each test vessel at the end of the test.

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (1200 µg/L nominal DMBPC concentration) was combined in a 250 mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 48 hours.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1 165 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
cell number
Remarks on result:
other: 95 % CL = 1029 to >1165 µg/L
Duration:
96 h
Dose descriptor:
EC90
Effect conc.:
> 1 165 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
cell number
Remarks on result:
other: 95 % CL = 1094 to >1165 µg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1 165 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL > 1165 µg/L
Duration:
96 h
Dose descriptor:
EC90
Effect conc.:
> 1 165 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL .1165 µg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1 141 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: area under the curve
Remarks on result:
other: 95 % CL = 837 to >1165 µg/L
Duration:
96 h
Dose descriptor:
EC90
Effect conc.:
> 1 165 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: area under the curve
Remarks on result:
other: 95 % CL > 1165 µg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
595 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
161 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: area under the curve
Details on results:
Insoluble material was not observed during the test.
The initial measured concentrations ranged from 97 to 108 % of the nominal concentrations and the final measured concentrations were 57 to 76 % of the nominal concentrations.

The algal population grew well, resulting in an average of 4 173 000 cells/mL in the control after 96 hours. No effects (relative size differences, unusual cell shapes, colours, 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. The incubator temperature ranged from 24.3 to 24.7 °C. The pH was not affected by the test substance.

Exposure of Selenastrum capricornutum to DMBPC for 96 hours resulted in an EC50 greater than 1165 µg/L when calculated using the number of cells/mL or the average specific growth rate, and 1141 µg/L when calculated using the area under the growth curve. The 96 hour NOEC is 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate, at 161 µg/L when determined using the area under the growth curve.

At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (the 1200 g/L) was combined with 100 mL of fresh media in a 250 mL flask. This culture was incubated under test conditions for 48 hours. During this period the number of algal cells increased from an initial calculated concentration of approximately 36 000 cells/mL to 676 000 cells/mL, indicated that any toxic effect at this concentration was algistatic rather than algicidal.
Reported statistics and error estimates:
The average specific growth rate was calculated as the natural log of the number of cells/mL at time t1 minus the natural log of the number of cells/mL at time t0 divided by the exposure period. The area under the growth curve was calculated as:

Area = (N1 - N0) / 2) x [(t1 + (N1 + N2 - 2N0) / 2)] x [(t2 - t1) + (Nn - 1 + Nn - 2N0) / 2] x (tn - tn - 1);

where: N0 = nominal number of cells/mL at time t0, N1 = measured number of cells/mL at time t1, Nn = measured number of cells/mL at time tn, t1 = time of first measurement after the beginning of the test and tn = time of nth measurement after the beginning of the test.

The EC10, EC50 and EC90 values were calculated using a weighted least squares non linear regression technique when warranted.

The no observed effect concentration (NOEC) was determined using a one-way analysis of variance (ANOVA) and Bonferroni’s test (TOXSTAT 3.3). The effective concentrations and NOECs were determined using the initial measured concentrations of DMBPC and the number of cells/mL, average specific growth rate, and area under the growth curve. Treatment data were compared to pooled control and solvent control data for the determination of the NOECs (mean control and solvent control data were never significantly different).

Effective concentrations (EC10, EC50 and EC90 values) from the toxicity test with DMBPC and the freshwater alga, Selenastrum capricornutum























































































































































































































































































Duration



Endpoint



Effect conc.


(microg/L)



Nominal/ Measured



Conc. Based on (nominal, a.i., test material)



Basis for Effect



95% CL


(microg/L)



48 hours



EC10



178



measured



a.i.



Cell number



<81 to 505



48 hours



EC50



>1165



measured



a.i.



Cell number



800 to >1165



48 hours



EC90



>1165



measured



a.i.



Cell number



>1165



72 hours



EC10



--



measured



a.i.



Cell number



--



72 hours



EC50



530



measured



a.i.



Cell number



248 to 1130



72 hours



EC90



>1165



measured



a.i.



Cell number



947 to >1165



96 hours



EC10



--



measured



a.i.



Cell number



--



96 hours



EC50



>1165



measured



a.i.



Cell number



1029 to >1165



96 hours



EC90



>1165



measured



a.i.



Cell number



1094 to >1165



 



 



 



 



 



 



 



48 hours



EC10



>1165



measured



a.i.



Growth rate



--



48 hours



EC50



>1165



measured



a.i.



Growth rate



>1165



48 hours



EC90



>1165



measured



a.i.



Growth rate



>1165



72 hours



EC10



>1165



measured



a.i.



Growth rate



--



72 hours



EC50



>1165



measured



a.i.



Growth rate



>1165



72 hours



EC90



>1165



measured



a.i.



Growth rate



>1165



96 hours



EC10



>1165



measured



a.i.



Growth rate



--



96 hours



EC50



>1165



measured



a.i.



Growth rate



>1165



96 hours



EC90



>1165



measured



a.i.



Growth rate



>1165



 



 



 



 



 



 



 



48 hours



EC10



--



measured



a.i.



AUC



--



48 hours



EC50



>1165



measured



a.i.



AUC



930 to >1165



48 hours



EC90



>1165



measured



a.i.



AUC



>1165



72 hours



EC10



--



measured



a.i.



AUC



--



72 hours



EC50



653



measured



a.i.



AUC



360 to >1165



72 hours



EC90



>1165



measured



a.i.



AUC



>1165



96 hours



EC10



--



measured



a.i.



AUC



--



96 hours



EC50



1141



measured



a.i.



AUC



837 to >1165



96 hours



EC90



>1165



measured



a.i.



AUC



>1165



AUC = Area under the curve



 


 


 


 


Measured concentrations of DMBPC in test media during the toxicity test with the freshwater alga,  Selenastrum capricornutum.





















































































































Nominal Concentration of DMBPC (microg/L)



Measured Concentrations of DMBPC
(microg/L)



0 Hour



% Recovery



96 Hours



% Recovery



Test Media Samples



Control



ND



--



ND



--



Solvent Control



ND



--



ND



--



75



81



108



43



57



150



161



107



98



65



300



297



99



228



76



600



595



99



435



73



1200



1165



97



900



75



Laboratory Control Sample



300



311



104



317



106



Matrix Spike Blank



300



--



--



285



95



--



--



277



92



Stability Blank



1200



1181



98



1036



86



Blank



0



ND



--



ND



--



ND = none detected at or above 8.0 microg/L, the limit of detection.



 


 


 


 


Cell growth data from the toxicity test with DMBPC and the freshwater alga,  Selenastrum capricornutum.















































































































































































































































































































Initial Measured


Concentration of


DMBPC


(microg/L)



Rep



Number of Cells per Milliliter



Hour of Exposure



0



24



48



72



96



Control



1



10,000



37,000



174,000



1,290,000



4,920,000



2



10,000



36,000



150,000



2,020,000



3,740,000



3



10,000



29,000



170,000



1,220,000



3,860,000



Mean



10,000



34,000



165,000



1,510,000



4,173,000



Solvent Control



1



10,000



34,000



136,000



880,000



4,420,000



2



10,000



45,000



130,000



700,000



4,820,000



3



10,000



35,000



156,000



960,000



4,880,000



mean



10,000



38,000



141,000



847,000



4,707,000



% control



100



112



85



56



113



81



1



10,000



43,000



146,000



840,000



5,360,000



2



10,000



31,000



188,000



860,000



3,220,000



3



10,000



23,000



140,000



720,000



4,100,000



mean



10,000



32,000



158,000



807,000



4,227,000



% control



100



94



96



53



101



161



1



10,000



24,000



146,000



772,000



4,640,000



2



10,000



47,000



142,000



1,260,000



4,720,000



3



10,000



36,000



150,000



720,000



4,400,000



mean



10,000



36,000



146,000



917,000



4,587,000



% control



100



106



88



61



110



297



1



10,000



26,000



124,000



720,000



3,440,000



2



10,000



49,000



126,000



780,000



3,760,000



3



10,000



47,000



150,000



940,000



3,720,000



mean



10,000



41,000



133,000



813,000



3,640,000



% control



100



121



81



54



87



595



1



10,000



36,000



100,000



448,000



3,820,000



2



10,000



43,000



116,000



830,000



4,660,000



3



10,000



40,000



66,000



430,000



3,640,000



mean



10,000



40,000



94,000



569,000



4,040,000



% control



100



118



57



38



97



1165



1



10,000



24,000



82,000



304,000



2,280,000



2



10,000



23,000



98,000



356,000



2,860,000



3



10,000



25,000



76,000



294,000



2,140,000



mean



10,000



24,000



85,000



318,000



2,427,000



% control



100



71



52



21



58



Rep = Replicate



 


 


 


 


Average specific growth rate from the toxicity test with DMBPC and the freshwater alga,  Selenastrum capricornutum.






















































































































Measured


Concentration


of DMBPC


(microg/L)



 



Average Specific Growth Rate



Hour of Exposure



24



48



72



96



Control



mean



0.051



0.058



0.069



0.063



Solvent Control



mean



0.055



0.055



0.062



0.064



% control



108



95



90



102



81



mean



0.048



0.057



0.061



0.063



% control



94



98



88



100



161



mean



0.051



0.056



0.062



0.064



% control



100



97



90



102



297



mean



0.057



0.054



0.061



0.061



% control



112



93



88



97



595



mean



0.057



0.046



0.056



0.062



% control



112



79



81



98



1165



mean



0.037



0.045



0.048



0.057



% control



73



78



70



90



Limit of detection was 8.0 microg/L


 


 


 


Area under the growth curve from the toxicity test with DMBPC and the freshwater alga,  Selenastrum capricornutum.


 






















































































































Measured


Concentration


of DMBPC


(microg/L)



 



Area Under the Growth Curve



Hour of Exposure



24



48



72



96



Control



mean



288,000



2,436,000



22,296,000



90,252,000



Solvent Control



mean



336,000



2,244,000



14,100,000



80,268,000



% control



117



92



63



89



81



mean



264,000



2,304,000



13,644,000



73,812,000



% control



92



95



61



82



161



mean



312,000



2,256,000



14,772,000



80,580,000



% control



108



93



60



89



297



mean



372,000



2,220,000



13,332,000



66,528,000



% control



129



91



60



74



595



mean



360,000



1,728,000



9,440,000



64,512,000



% control



125



71



42



71



1165



mean



168,000



1,236,000



5,832,000



38,532,000



% control



58



51



26



43


Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 96 hour ErC50 was > 1165 µg/L. The 96 hour NOEC was 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate.
Executive summary:

The toxicity potential of the test material to freshwater algae was investigated in accordance with the standardised guideline EPA OPPTS 850.5400 under GLP conditions.

The definitive toxicity test was conducted at 24 ± 2°C with five concentrations of test substance, a control, and a solvent control (0.10 mL/L dimethylformamide). Nominal concentrations were 0 (control and solvent control), 75, 150, 300, 600 and 1200 µg/L. Each solution was subdivided into three clear glass 250 mL Erlenmeyer flasks. The flasks were inoculated with approximately 10 000 algal cells/mL and capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator. 

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer at 24, 48, 72, and 96 hours. 

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (1200 µg/L nominal DMBPC concentration) was combined in a 250 mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 48 hours.

Insoluble material was not observed during the test.  The initial measured concentrations ranged from 97 to 108 % of the nominal concentrations and the final measured concentrations were 57 to 76 % of the nominal concentrations.

The algal population grew well, resulting in an average of 4 173 000 cells/mL in the control after 96 hours. No effects were noted during the test. The 0.5 mL aliquot that was incubated for a further 48 hours indicated that any toxic effect at this concentration was algistatic rather than algicidal.

Exposure of algae to DMBPC for 96 hours resulted in a median effective concentration (EC50) that was greater than 1165 µg/L (the highest tested concentration and reported water solubility limit) when calculated using the number of cells/mL or the average specific growth rate, and 1141 µg/L when calculated using the area under the growth curve. The 96 hour no observed effect concentration (NOEC) is 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate, and 161 µg/L when determined using the area under the growth curve.

Description of key information

Under the conditions of this study, the 96 hour ErC50 was > 1165 µg/L. The 96 hour NOEC was 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
595 µg/L

Additional information

The toxicity potential of the test material to freshwater algae was investigated in accordance with the standardised guideline EPA OPPTS 850.5400 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The definitive toxicity test was conducted at 24 ± 2°C with five concentrations of test substance, a control, and a solvent control (0.10 mL/L dimethylformamide). Nominal concentrations were 0 (control and solvent control), 75, 150, 300, 600 and 1200 µg/L. Each solution was subdivided into three clear glass 250 mL Erlenmeyer flasks. The flasks were inoculated with approximately 10 000 algal cells/mL and capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator. 

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer at 24, 48, 72, and 96 hours. 

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (1200 µg/L nominal DMBPC concentration) was combined in a 250 mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. This culture was incubated under test conditions for 48 hours.

Insoluble material was not observed during the test.  The initial measured concentrations ranged from 97 to 108 % of the nominal concentrations and the final measured concentrations were 57 to 76 % of the nominal concentrations.

The algal population grew well, resulting in an average of 4 173 000 cells/mL in the control after 96 hours. No effects were noted during the test. The 0.5 mL aliquot that was incubated for a further 48 hours indicated that any toxic effect at this concentration was algistatic rather than algicidal.

Exposure of algae to DMBPC for 96 hours resulted in a median effective concentration (EC50) that was greater than 1165 µg/L (the highest tested concentration and reported water solubility limit) when calculated using the number of cells/mL or the average specific growth rate, and 1141 µg/L when calculated using the area under the growth curve. The 96 hour no observed effect concentration (NOEC) is 595 µg/L DMBPC when determined using the number of cells/mL or the average specific growth rate, and 161 µg/L when determined using the area under the growth curve.