2,5-di-tert-pentylhydroquinone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 September 2016 to 20 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C3: “Algal Inhibition Test”; Amended by EC No. 2016/266 of 7 December 2015, Publication No. L54.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2.0 mL
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

not specified

Details on test solutions:

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.1% and the item was not completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 10 mg/L applying 3 days of magnetic stirring to ensure maximum dissolution. The resulting dispersion was filtered through a 0.45 µm membrane filter (Whatman RC55) to remove the undissolved fraction of test item. The resulting clear and colourless Saturated Solution (SS) was used as highest test concentration and used to prepare a range of dilutions for the lower test concentrations. All final test solutions were clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of e+4 cells/mL.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh water algae culture
Stock culture : Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit).

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations: 4.6, 10, 22, 46 and 100% of a SS prepared at a loading rate of 10 mg/L

Details on test conditions:

Range-finding test
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
-Exponentially growing algal cultures were exposed to 1.0, 10 and 100% of a SS prepared at a loading rate of 10 mg/L and to a control.
-Three replicates were tested per concentration and three replicates in the control group.
-Cell densities were only recorded at the end of the exposure period.
-pH was only measured in the control and the highest test concentration.
-At the end of the test algae were not observed to verify a normal and healthy appearance.
-No sampling for determination of actual test concentrations was performed.

Solubility test using algal growth medium
After the range-finding test a solubility test was performed to confirm the expectation that the solubility in test medium was much lower than the solubility in pure water that was indicated to be 1 g/L at 20°C. The column elution method was used to determine the medium solubility.

Final test
Test concentrations
Lowinox® AH25: 4.6, 10, 22, 46 and 100% of a SS prepared at a loading rate of 10 mg/L
Controls: Test medium without test item or other additives
Replicates: 3 replicates of each test concentration,
6 replicates of the control,
1 or 2 replicates of each test concentration without algae,
1 extra replicate of each test group with algae for sampling after 24 hours.

Test procedures and conditions
Test duration :72 hours
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1e+4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 96 to 99 µE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements
pH: At the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test microscopic observations were performed on the two highest test concentrations and the control to observe for any abnormal appearance of the algae.

Recording of cell densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates incubated without algae as background for the treated solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

49 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

246 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Details on results:

Final test
Growth rates were in the range of the controls at TWA concentrations up to 12 µg/L during the 72-hour test period, whereas the growth rate of algae exposed to 49 and 130 µg/L were increasingly reduced.
Statistically significant inhibition of growth rate was found at test concentrations of 49 µg/L and higher. However, since the inhibition of 8.5% at 49 µg/L was not considered biologically relevant, the NOEC was set at 49 µg/L.
Inhibition of yield ranged between -2.9 and 11.9% at TWA concentrations of 0.4 to 12 µg/L. At 49 and 130 µg/L the inhibition was significantly higher with 35 and 78%. Statistically significant inhibition of yield was found at TWA concentrations of 49 µg/L and higher.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 49 and 130 µg/L when compared to the control.

Results with reference substance (positive control):

Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.32 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.2 to 1.4 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.45 mg/L with a 95% confidence interval ranging from 0.39 to 0.53 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Mean cell densities (x10⁴cells/mL) during the range-finding test

Time (h)	Lowinox® AH25 (%SS of 10 mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	nm	nm	nm	nm
48	nm	nm	nm	nm
72	117.4	118.3	134.1	51.4

nm: not measured

 

Percentage inhibition of growth rate during the range-finding test

Lowinox® AH25 (%SS of 10 mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.586	0.0526	3
1.0	1.590	0.0370	3	-0.2
10	1.633	0.0173	3	-2.9
100	1.313	0.0193	3	17.2

 

Percentage inhibition of yield during the range-finding test

Lowinox® AH25 (%SS of 10 mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	116.4	19.13	3
1.0	117.3	13.36	3	-0.7
10	133.1	6.86	3	-14.3
100	50.4	3.03	3	56.7

 

Measured and average measured concentrations

Lowinox® AH25 (%SS of 10 mg/L)	Measured concentration (mg/L)			TWA (μg/L)
	t=0h	t=24h	t=72h*
4.6	0.00669	0.000174	0.00000225	0.4
10	0.0334	0.00102	0.00000225	2.0
22	0.133	0.0103	0.00000225	12
46	0.395	0.0534	0.00000225	49
100	1.09	0.138	0.00000225	130

* Concentration sex at 0.5 x LOD.

 

Percentage inhibition of growth rate (total test period) during the final test

Lowinox® AH 25 TWA conc. (μg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.659	0.0378	6
0.4	1.615	0.0561	3	2.6
2.0	1.669	0.0326	3	-0.6
12	1.631	0.0261	3	1.7
49	1.517	0.0238	3	8.5*/^#
130	1.165	0.0399	3	29.7*

* - effect was statistically significant

^#- effect biologically irrelevant (<10%)

 

Percentage inhibition of growth rate at different time intervals during the final test

Lowinox® AH25 TWA conc. (μg/L)	n	0 – 24 h		24 – 48 h		48 – 72 h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.566	0.0	1.801	0.0	1.609	0.0
0.4	3	1.560	0.3	1.675	7.0	1.610	-0.1
2.0	3	1.596	-1.9	1.761	2.2	1.649	-2.5
12	3	1.556	0.6	1.684	6.5	1.652	-2.7
49	3	1.426	8.9	1.549	14.0	1.576	2.0
130	3	1.150	26.5	1.019	43.4	1.327	17.5

 

Percentage inhibition of yield during the final test

Lowinox® AH 25 TWA conc. (μg/L)	Mean	Std. Dev.	n	%Inhibition
Control	144.6	16.05	6
0.4	127.4	20.97	3	11.9
2.0	148.7	14.95	3	-2.9
12	132.5	10.56	3	8.4
49	93.9	6.78	3	35.1*
130	32.1	4.06	3	77.8*

* - effect was statistically significant

 

Overview of % inhibition of growth rate in the reference test:

Nominal conc. K2Cr2O7(mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.475	0.0948	3
0.18	1.421	0.1100	3	3.7
0.32	1.182	0.3745	3	19.9
0.56	1.221	0.0545	3	17.2
1.0	1.080	0.0239	3	26.8
1.8	0.432	0.0521	3	70.7
3.2	0.227	0.0183	3	84.6

 

Overview of % inhibition of yield in the reference test:

Nominal conc. K2Cr2O7(mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	84.8	23.34	3
0.18	72.5	21.61	3	14.5
0.32	46.8	35.15	3	44.8
0.56	38.3	6.67	3	54.8
1.0	24.6	1.81	3	71.0
1.8	2.7	0.56	3	96.8
3.2	1.0	0.11	3	98.8

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Lowinox® AH25 reduced growth rate of this fresh water algae species significantly at a TWA concentration of 130 µg/L.
The EC50 for growth rate inhibition (72h-ERC50) was 246 µg/L with a 95% confidence interval ranging from 209 to 313 µg/L.
The EC50 for yield inhibition (72h-EYC50) was 66 µg/L with a 95% confidence interval ranging from 55 to 79 µg/L.
The 72h-NOEC for growth rate inhibition was 49 µg/L, while the NOEC for yield inhibition was 12 µg/L, both based on TWA concentrations.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Lowinox® AH25.

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 2016/266 and the OECD series on testing and assessment number 23, 2000.

 

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.1% and the item was not completely soluble in test medium at the concentrations tested. Preparation of test solutions started with a loading rate of 10 mg/L applying 3 days of magnetic stirring followed by filtration through a 0.45 µm membrane filter to remove the undissolved fraction of test item. The resulting clear and colourless Saturated Solution (SS) was used as highest test concentration and used to prepare a range of dilutions for the lower test concentrations.

 

A final EC50 test was performed based on the results of a preceding range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to test groups containing 4.6, 10, 22, 46 and 100% of a SS prepared at a loading rate of 10 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

 

Analyses showed that the actual test concentration at the start of the test in the saturated solution (SS) prepared at 10 mg/l was 1.09 mg/L. The concentrations measured in the dilutions were more or less in agreement with the respective dilution factors. The concentrations measured after 24 hours all decreased significantly to 2.6-14% of the initial measured concentrations. All concentrations had decreased below the Limit Of Detection (LOD <0.0045 µg/L) at the end of the test. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated to correspond with 0.4, 2.0, 12, 49 and 130 µg/L.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

CONCLUSION

Under the conditions of the present study with Pseudokirchneriella subcapitata, Lowinox® AH25 reduced growth rate of this fresh water algae species significantly at a TWA concentration of 130 µg/L.

 

The EC50 for growth rate inhibition (72h-ERC50) was 246 µg/L with a 95% confidence interval ranging from 209 to 313 µg/L.

 

The EC50 for yield inhibition (72h-EYC50) was 66 µg/L with a 95% confidence interval ranging from 55 to 79 µg/L.

 

The 72h-NOEC for growth rate inhibition was 49 µg/L, while the NOEC for yield inhibition was 12 µg/L, both based on TWA concentrations.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1980-04-14 to 1980-04-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, sufficiently well-documented study which meets basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A phytotoxicity test was performed by exposing freshwater algae to a series of test concentrations of Santovar A. Chlorophyll alpha content was reported after 24, 48, 72 and 96 hours, and cell counts were reported after 96 hours. Culture and test procedures followed those of US EPA (1978) The Selenastrum capricornutum printz algal assay test. (Corvallis Environmental Research Laboratory, Oregon, USA).

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

no data

Vehicle:

yes

Details on test solutions:

- vehicle: triethylene glycol (TEG)
- a primary stock solution was prepared by adding a weighed amount of test material to reagent grade triethylene glycol (TEG)
- a secondary stock solution was prepared by serial dilution of the primary stock, for the range-finding and the definitive test
- test solutions were prepared by adding appropriate amounts of stock solutions to each flask

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- common name: green alga (unicellular)
- source: culture obtained from the U.S. Environmental Protection Agency's Environmental Research Laboratory, Corvallis, Oregon and maintained in stock culture at BMRL
- medium: AAP freshwater medium
- inoculum culture was maintained in nutritionally dilute culture medium during 7 days prior to testing

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

no data

Test temperature:

24 ± 1 °C

pH:

- at time 0: 7.2 in all treatments
- after 96 h: 7.6 in 0.3 mg/L, 7.6 in 0.6 mg/L, 7.4 in 1.2 mg/L, 7.1 in 2.5 mg/L, 7.0 in 5 mg/L and 6.9 in 10 mg/L

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

nominal concentrations: control, solvent control, 0.3, 0.6, 1.2, 2.5, 5.0, 10 mg/L

Details on test conditions:

- illumination: 4000 lux for 24h/d
- n° replicates: 3 for each test concentration and each control
- solvent control: 0.05 mL TEG, which is the maximum volume added to the test flasks
- light setting: 24 hour light
- test container volume: 125 mL
- test medium volume: 50 mL
- initial cell-count: 2.0 x 10^4 cells/mL
- volume of inoculum added to each test flask: 1 mL
- range-finding test: 72-h exposure
* control, 0.1, 1, 10 and 100 mg/L
* pH after 72 h: 7.0 in control, 7.1 in 0.1 mg/L, 7.1 in 10 mg/L and 7.0 in 100 mg/L
- in vivo chlorophyll alfa results: reported but units are not given so data are not copied in this section. Overall, the chlorophyll alfa content decreased with increasing exposure concentration with a clear effect in 10 and 100 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: in vivo chlorophyll alfa content

Remarks on result:

other: 95 % CL: 0.2-18 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95 % CL: 1.7-4.8 mg/L

Details on results:

- there was no significant difference between growth of the control and solvent control cultures after 96 hours of exposure, based on both in vivo chlorophyll alfa and cell numbers
- after 96 hours, chlorophyll alfa decreased with 4 % in cultures exposed to 0.6 mg/L and 89 % in those exposed to 10 mg/L
- after 96 hours, chlorophyll alfa increased in cultures exposed to less than 0.3 mg/L
- after 96 hours, cell numbers decreased with 8 % in cultures exposed to 1.2 mg/L and 82 % in those exposed to 10 mg/L
- after 96 hours, cell numbers increased in cultures exposed to less than 0.6 mg/L
- other effect concentrations for in vivo chlorophyll A content: 24h-EC50 >10 mg/L; 48h- and 72h-EC50 1.2-2.5 mg/L

Reported statistics and error estimates:

- each test concentration was converted to a logarithm and the corresponding percentage decrease of in vivo chlorophyll alfa or cell numbers was converted to a probit (Finney, 1971)
- both of the 96-h EC50 values and their 95 % confidence limits were then calculated by linear regression
- differences in growth between the solvent control and the culture medium control were determined by the Student's T-test (Steel and Torrie, 1960). Differences were considered significant at the 95 % confidence level.

Validity criteria fulfilled:

not applicable

Conclusions:

Reliable study (Klimisch 2) reporting a 96-h EC50 value of 1.7 mg/L for in vivo chlorophyll a and 2.9 mg/L for cell number. Although not performed following the OECD guidelines, the study was judged to be adequately performed and sufficiently documented.

Executive summary:

A phytotoxicity test was performed at EG&G, Bionomics Marine Research Laboratory (BMRL), Pensacola, Florida, to determine the effect of Santovar A on the freshwater alga Selenastrum capricornutum. Results of the test are reported as 24-, 48-, 72-, and 96-hour EC50's (the concentrations of the test material estimated to cause a 50% decrease of in vivo chlorophyll a in exposed cultures as compared to the control at the specified times). Cell numbers in exposed and control cultures were also determined after 96 hours of exposure and another 96-hour EC50 was calculated (the concentration of the test material estimated to cause a 50% decrease of cell numbers in exposed cultures as compared to the control). Confirmation of effect by measurement of two different growth factors is important, in our opinion, because of the various alga responses in the presence of toxicants (Hall, 1973).

 

Concentrations of Santovar A ≥ 2.5 ppm appeared to adversely affect growth of Selenastrum capricornutum throughout the 96 hours of exposure. Based on decrease of in vivo chlorophyll a, the estimated 24-hour EC50 was >10 ppm while the calculated 96-hour EC50 was 1.7 ppm with 95% confidence limits of 0.2-18. ppm.

The calculated 96-hour EC50, based on cell number decrease, was 2.9 ppm with 95% confidence limits of 1.7-4.8 ppm.

After 96 hours of exposure, decrease of in vivo chlorophyll a was from 4% in cultures exposed to 0.6 ppm to 89% in those exposed to 10 ppm; in vivo chlorophyll a increased in cultures exposed to ≤ 0.3 ppm Decrease of cell numbers was from 8% in cultures exposed to 1.2 ppm to 82% in those exposed to 10 ppm; cell numbers increased in cultures exposed to ≤ 0.6 ppm after 96 hours. The pH was from 6.9-7.6 after 96 hours of exposure.

There was no significant difference between growth of the control and solvent control cultures after 96 hours of exposure, based on both in vivo chlorophyll a and cell numbers.

Description of key information

Key study

The 72h-NOEC for growth rate inhibition was 49 µg/L, while the NOEC for yield inhibition was 12 µg/L, both based on TWA concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

66 µg/L

EC10 or NOEC for freshwater algae:

12 µg/L

Additional information

Key Study

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.1% and the item was not completely soluble in test medium at the concentrations tested. Preparation of test solutions started with a loading rate of 10 mg/L applying 3 days of magnetic stirring followed by filtration through a 0.45 µm membrane filter to remove the undissolved fraction of test item. The resulting clear and colourless Saturated Solution (SS) was used as highest test concentration and used to prepare a range of dilutions for the lower test concentrations. 

A final EC50 test was performed based on the results of a preceding range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to test groups containing 4.6, 10, 22, 46 and 100% of a SS prepared at a loading rate of 10 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

CONCLUSION

Under the conditions of the present study with Pseudokirchneriella subcapitata, Lowinox® AH25 reduced growth rate of this fresh water algae species significantly at a TWA concentration of 130 µg/L.

The EC50 for growth rate inhibition (72h-ERC50) was 246 µg/L with a 95% confidence interval ranging from 209 to 313 µg/L.

The EC50 for yield inhibition (72h-EYC50) was 66 µg/L with a 95% confidence interval ranging from 55 to 79 µg/L.

The 72h-NOEC for growth rate inhibition was 49 µg/L, while the NOEC for yield inhibition was 12 µg/L, both based on TWA concentrations.

Supporting study

The freshwater green alga Selenastrum capricornutum was exposed to Santovar A during 96 hours in a static test. Based on the cell numbers, an EC50 value of 2.9 mg/L was derived.