N,N,4-trimethylpiperazine-1-ethylamine

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:

2017-06-16 to 2017-11-11

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC No. 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW160141
- Expiration date of the lot/batch: 2018-12-30
- Purity test date: 2016-07-19 (certificate of analysis release date)
- Purity: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
* Range-Finding Test: A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.
* Definitive Test: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
* Range-Finding Test: The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity
* Definitive Test: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L. An initial test conducted showed inconsistent results from chemical analysis and hence has not been reported. An amount of test item (101.1 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a nominal 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.9 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): solutions were clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4.
- Source: Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1”C.
- Pre-culture: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1”C until the algal cell density was approximately 10^4 – 10^5 cells/mL.

ACCLIMATATION
- Acclimatation period: not relevant

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

Temperature was maintained at 24 ± 1 °C

pH:

The pH value of the control cultures was observed to range from pH 7.5 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours showed measured test concentrations of 126%, 125% and 104% of nominal respectively were obtained. Analysis of the test preparations at 72 hours showed no significant decline in measured concentrations occurred with the exception of the 100 mg/L test sample where a measured concentration of 65% of nominal was obtained.

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: 250 mL glass conical flasks each containing 100 mL of test preparation
- Initial cells density: 5.00 x 10^3 cells per mL
- Control end cells density: 1.14 x 10^6 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Culture Medium
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: the pH was adjusted to 7.5 with 0.1N NaOH or HCl
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Samples were taken at 25, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample.
- Effect calculated parameters: specific growth rate and yield.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 3.2
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/L, however misshapen cells were observed to be present in the test cultures at 100 mg/L.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/L test cultures were observed to be green dispersions whilst the 32 and 100 mg/L test cultures were observed to be clear colorless solutions
- Effect concentrations exceeding solubility of substance in test medium:
- ErC10 (0 - 72 h): 9.7 mg/L
- ErC20 (0 - 72 h): 15 mg/L
- ErC50 (0 - 72 h): 29 mg/L; 95% confidence limits 23 - 37 mg/L
- EyC10 (0 - 72 h): 5.5 mg/L
- EyC20 (0 - 72 h): 7.2 mg/L
- EyC50 (0 - 72 h): 12 mg/L; 95% confidence limits 9.9 - 14 mg/L
- The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
- The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
- ErC50 (0 – 72 h): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
- EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L
- No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
- No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
- Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
- Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
- The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

- Statistical analysis of the growth rate data was carried out for the control and all test concentrations using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P0.05), between the control, 1.0 and 3.2 mg/L test groups and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/L.
- Statistical analysis of the yield data was carried out as above. There were no statistically significant differences (P0.05), between the control, 1.0 and 3.2 mg/L test groups and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/L.

Table 1: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.077		1.30E+06
	R2	0.074		1.02E+06
	R3	0.073		9.86E+05
	R4	0.075	-	1.12E+06	-
	R5	0.076		1.19E+06
	R6	0.076		1.18E+06
	Mean	0.075		1.13E+06
	SD	0.001		1.17E+05
1.0	R1	0.080	[7]	1.54E+06
	R2	0.077	[3]	1.31E+06
	R3	0.075	0	1.09E+06
	Mean	0.077	[3]	1.32E+06	[16]
	SD	0.003		2.25E+05
3.2	R1	0.077	[3]	1.25E+06
	R2	0.077	[3]	1.24E+06
	R3	0.069	8	7.25E+05
	Mean	0.074	1	1.07E+06	5
	SD	0.005		3.02E+05
10	R1	0.070	7	7.57E+05
	R2	0.069	8	7.13E+05
	R3	0.067	11	6.09E+05
	Mean	0.069	9	6.93E+05	39
	SD	0.002		7.57E+04
32	R1	0.036	52	6.01E+04
	R2	0.031	59	4.06E+04
	R3	0.032	57	4.55E+04
	Mean	0.033	56	4.87E+04	96
	SD	0.003		1.01E+04
100	R1	0.012	84	6.90E+03
	R2	0.007	91	3.24E+03
	R3	0.005	93	2.23E+03
	Mean	0.008	89	4.13E+03	100
	SD	0.004		2.46E+03

*       In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Remarks:

The data satisfied the validation criterion given in the OECD Guideline

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance JeffCat TAP according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had no significant inhibitory effect on the growth of Pseudokirchneriella subcapitata up to and including the test concentration of 3.2 mg/L, after the test period of 72 hours. The nominal EC50 for growth rate inhibition (72-h ErC50) was 29 mg/L with a 95% confidence interval ranging from 23 to 37 mg/L. The results of the test can be considered reliable without restrictions.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	29	23	-	37	3.2	10
Yield	12	9.9	-	14	3.2	10

 

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001-11-14 to 2002-05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity: 99.2% (GC)
- Source and lot/batch No.of test material: 180601
- Expiration date of the lot/batch: 2002-10-25 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: at least 96h in water

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preperation of test solutions started with a stock solution of 100 mg/L applying 10 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the still stirring stock solution in test medium.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspention were added to each replicate providing a cell density of 10^4 cells/mL
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All solutions were clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Method of cultivation: 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 (4000-9000 lux) in a climate room at 23 ± 2°C.
- Pre-culture: 3 days before the start of the test , cells from the algal stock culture were inoculated in culture medium at a cell density of 2.10^4 cells/mL. the pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

ACCLIMATATION
- Acclimatation period: not relevant

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

22-23°C

pH:

at 0h: 8.6-9.1
at 72h: 8.0-8.7

Dissolved oxygen:

not reported

Salinity:

not relevant

Conductivity:

not relevant

Nominal and measured concentrations:

Range finder:
Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L

Final test:
Nominal concentrations: 10, 2.2, 4.6, 10, 22, 46 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass
- Material, size, headspace, fill volume: 100 mL glass
- Initial cells density: 1.00 x 10^4 cells per mL
- Control end cells density: 116.1 x 10^4 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Culture Medium
NaHCO3 50 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
K2HPO4 1.6 mg/L
NaHCO3 50 mg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
FeCl3.6H2O 80 µg/L
CoCl2.6H2O 1.5 µg/L
Na2MoO4.2H2O 7 µg/L
CuCl2.2H2O 0.01 µg/L
Na2EDTA.2H2O 100 µg/L
NH4Cl 15 mg/L
Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 7400 to 9600 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning of the test, cells were counted by microscope, using a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength = 20 nm).
- Effect calculated parameters: specific growth rate and yield.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 30 - 43 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- EbC10 (0 - 72 h): 6.4 mg/L (95% C.L.: 2.0 - 20.0 mg/L)
- ErC10 (0 - 72 h): 11.0 mg/L (95% C.L.: 9.1 - 13 mg/L)
- EbC50 (0 - 72 h): 20.0 mg/L (95% C.L.: 7.1 - 55.0 mg/L)

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- A positive control (Notox project 341685) used potassium dichromate as the reference item at concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Selenastrum capricornutum to the reference item gave the following results:
- EbC50 (0 – 72 h): 0.83 mg/L; 95% confidence limits 0.59 – 1.2 mg/L
- ErC50 (0 – 72 h): 0.16 mg/L; 95% confidence limits 1.3 –2.0 mg/L
- The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Calculation of the EC50 and EC10 values was based on linear regression analysis of the percentages of growth inhibition and the percentages of growth rate reduction versus the logarithms of the corresponding nominal concentrations of the test substance.

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Selenastrum capricornutum was performed with the test substance JeffCat TAP according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had a significant inhibitory effect on the growth of Selenastrum capricornutum. The nominal EC50 for growth rate inhibition (72-h ErC50) was 36 mg/L with a 95% confidence interval ranging from 30 to 43 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

Two relevant studies were identified to cover this endpoint.

The studies of Migchielsen (2002) and Vryenhoef (2018), investigating the acute toxicity of the test substance to algae according to OECD guideline 201, were considered as high quality studies for endpoint coverage.

Migchielsen (2002) obtained a nominal EC50 for growth rate inhibition (72-h ErC50) of 36 mg/L with a 95% confidence interval ranging from 30 to 43 mg/L, the 72-h NOEC for growth rate was 4.6 mg/L.

In the study of Vryenhoef (2018), the 72 -h NOEC and 72-h EC50 for growth rate were determined to be 3.2 mg/L and 29 mg/L respectively. The latter study was considered for the PNEC derivation. The substance can be considered as harmful to algae.

 

Key value for chemical safety assessment

EC50 for freshwater algae:

29 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L