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Toxicity to aquatic algae and cyanobacteria

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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were collected of the Stock Solution (WAF) and of each treatment at the beginning and end of exposure for analysis.

Vehicle:

no

Details on test solutions:

Algae medium was prepared in accordance with OECD 201. Specifically, the AAP
medium specified in Annex 3 from that method was used. Algae medium was prepared
using deionized water (DI). Each of the stock solutions was prepared in DI (Table 2).
One ml of each stock was added to a flask and diluted to one liter with DI water. This
water was mixed on a magnetic stir plate and filtered through a 0.2 micron filter for
sanitization. This medium was used for all culturing and testing of algae for this study.

Component Concentration in Prepared Medium (mg/L)
NaHCO3 15.0
NaNO3 25.5
MgCl2-6(H2O) 12.16
CaCl-2(H2O) 4.41
MgSO4-7H2O 14.6
K2HPO4 1.044
FeCl3-6(H2O) 0.160
Na2EDTA-2(H2O) 0.300
H3BO3 0.186
MnCl2-4(H2O) 0.415
ZnCl2 0.00327
CoCl2-6(H2O) 0.00143
Na2MoO4-(H2O) 0.00726
CuCl2-2(H2O) 0.000012

A 5g/L stock solution was prepared by transferring 5 grams of neat product onto a tared weigh boat using a pipet. The stock solution was prepared by transferring the weighed product to a volumetric flask and bringing the volume up to 1 liter with dilution water. The dilution water used was algae medium (AAP). This is the same dilution water used for testing. This stock solution was mixed on a stir plate with a magnetic stir bar for 24 hours. The apparently soluble portion of the stock solution (after 24h mixing) was further diluted to prepare the nominal concentrations for the test.

Test organisms (species):

other: Raphidocelis subcapitata

Details on test organisms:

Algae (R. subcapitata) were used in this study. An algal starter culture was purchased from Aquatic Biosystems, Inc. (Fort Collins, Colorado) prior to testing. BMI cultures were inoculated at 104 cells/ml and allowed to incubate for 72 hours. At the 72-hour mark, cell counts were made so as to determine the growth acceptability of the culture. Appropriate growth rates were achieved.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Nominal and measured concentrations:

Summary of Measured Concentrations

Nominal Conc. (mg/L) 0 Hour Conc. (mg/L) 72 Hour Conc. (mg/L) Mean Conc. (mg/L) Percent Nominal
0 0 0 0 100
0.3125 1.0 0.8 0.9 NA *
0.625 1.6 1.6 1.6 NA *
1.25 2.1 2.3 2.2 NA *
2.5 3.6 2.6 3.1 124
5 6.0 5.4 5.7 114
* These concentrations were below the calculated Limit of Quantification (LOQ).

Details on test conditions:

Summary of Test Conditions for the Freshwater Algal Growth Inhibition Test
1. Test type: Static
2. Renewal of test solutions: Not Renewed
3. Temperature (°C): 22 + 2°C
4. Light quality: Full Spectrum
5. Light intensity: 60 - 100 μE/(m-2 s-1)
6. Photoperiod: Continuous
7. Size of test vessel: 250 ml
8. Volume of test solution: 100 ml
9. Cell Density at test initiation: 10 000 cells/ml
11. Number of replicates per concentration: 3
14. Aeration: None
15. Medium: See relevant section
16. Test duration: 72 hours
17. Test concentrations: Five product concentrations and a control
18. Dilution series: 0.1 dilution series for range-finding test
0.5 dilution series for definitive test
19. Endpoint: Growth Inhibition
20. Sampling and sample: Water Accommodated Fraction (WAF)
21. Test acceptability criterion: Biomass of control cultures increase by a factor of at
least 16. Section – by – section specific growth rate
coefficient of variation not more than 35%.
Coefficient of variation during the whole test period
not greater than 7% in the controls.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

IC10

Effect conc.:

0.446 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

2.766 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Validity of the Test
The biomass in the control cultures increased exponentially by a factor of at least 16 within the 72 hour testing period and had a specific growth rate greater than 0.92 per day.
The mean coefficient of variation for days 0-1 was 20.915%, for days 1-2 was 22.346%, and for days 2-3 it was 7.367%. None of these exceeded the method specified limit of 35%.
The coefficient of variation of average specific growth rate during the entire test in replicate control cultures was 2.779% and did not exceed the method specified limit of 7%.

Observation of abnormalities (for algal test):
The cells in all treatments and the control appeared normal at each interval of the test. No abnormalities.

Measured Concentrations
Sample recovery ranged from 114% (5 mg/L) of the nominal concentration to 124% (2.5 mg/L) of the nominal concentration. Lower concentrations were below the calculated Limit of Quantification (LOQ). Therefore, only the 2.5 and 5.0 mg/L concentrations were used to calculated percent recovery. Table above presents a summary of the measured concentration data. Since the measured concentrations (above the LOQ) had a mean within 20% of the nominal, nominal concentrations were used in determining the IC10 and IC50.

Growth Rate Results
Growth rates ranged from 0.0064 for the 5 mg/L treatment to 0.0623 for the control. Inhibition ranged from 1.16% in the 0.3125 mg/L treatment to 89.70% in the 5 mg/L treatment. Table 4 presents a summary of the growth and inhibition data.

Concentration-mg/L Mean Specific Growth Rate % Inhibition
0 0.0623 NA

0.3125 0.0616 1.16
0.625 0.0487 21.90
1.25 0.0400 35.78
2.5 0.0341 45.27
5 0.0064 89.70

The IC10 value calculated was 0.4457 mg/L for Benzylated Polyamine. The 95% confidence limits were 0.3445 and 0.6129 mg/L. The IC50 value calculated was 2.7662 mg/L. The 95% confidence limits were 2.5607 and 2.9628 mg/L. The Linear Interpolation method was used for this test.

results of algal biomass determined in each flask daily during the test period reported in a tabular form:

cell density at test initiation: 10.000 cells/ml

Nominal conc. (mg/L)	Rep.	24h (10^4 Cells/ml)	48h (10^4 Cells/ml)	72h (10^4 Cells/ml)	pH (SU)	Light Intensity
0	A	5	18	100	7.58/8.66	430-560
	B	4	17	78
	C	8	20	90
0.3125	A	3	20	89	7.62/8.62
	B	4	19	75
	C	2	19	90
0.625	A	3	18	29	7.74/8.16
	B	2	16	27
	C	2	19	47
1.25	A	1	12	19	7.79/7.88
	B	3	15	13
	C	3	17	23
2.5	A	3	14	12	7.85/7.71
	B	2	12	11
	C	3	15	12
5	A	0	10	2	7.84/7.56
	B	1	8	2
	C	0	8	1
10	A	0	3	0	7.75/7.51
	B	0	1	0
	C	0	1	0

 

Average area obtained for running standards (3 times) for area of three different peaks

Std’s	Conc., ug/mL	1-  4.2min	2- 5.92min	3- 9.3min	total
Std1	0.55	ND	1.4	1.7	3.1
Std2	1.1	1.3	2.6	3.5	7.5
Std3	5.5	9.7	16.9	6.7	33.3
Std4	10.9	19.5	32.3	12.3	64.2
Std5	27.5	52.4	86.7	28.6	167.8
Std6	54.8	107.0	173.1	52.3	332.5
Std7	109.6	200.7	351.2	106.9	658.9
Std8	219.2	422.1	700.6	203.0	1325.8
Std9	548.0	1049.0	1763.7	504.8	3317.6
Std10	1096.0	2132.3	3486.5	1033.7	6652.5

 

Calculated concentration of benzylated polyamine in different samples provided by BMI

	Sample	Conc., ug/mL	Conc., ug/mL	Conc., ug/mL	Conc., ug/mL
mg/L		Peak 1, 4.2 min	Peak 2, 5.92 min	Peak 3, 9.3 min	Total Areas
5000	EV-50001	2985.9	4990.3	4274.0	4237.8
5000	EV-50002	2860.0	4984.6	4177.8	4179.7
0	EV03-0hr-0mg/mL	0.0	0.0	0.0	0.0
0	EV03-72hr-0mg/mL	0.0	0.0	0.0	0.0
0.3125	EV03-0hr-0.3125mg/L	0.0	0.3	1.8	1.0
0.3125	EV03-72hr-0.3125mg/L	0.0	0.1	1.0	0.8
0.625	EV03-0hr-0.625mg/L	2.4	0.5	3.6	1.6
0.625	EV03-72-0.625mg/L	0.0	0.3	5.3	1.6
1.25	EV03-0hr-1.25mg/L	2.9	0.9	4.4	2.1
1.25	EV03-72hr-1.25mg/L	2.5	0.1	9.2	2.3
2.5	EV03-0hr-2.5mg/L	4.5	2.3	6.0	3.6
2.5	EV03-72-2.5mg/L	3.7	1.0	6.0	2.6
5	EV03-0hr-5mg/L	7.3	4.4	8.9	6.0
5	EV03-72hr-5mg/L	7.2	2.5	11.2	5.4
10	EV03-0hr-10mg/L	12.5	9.5	15.7	11.4
10	EV03-72hr-10mg/L	13.3	7.5	17.7	10.9
0	EV01-0hr-0mg/L	0.0	0.0	0.0	0.0
0	EVO1-96hr-0mg/L	0.0	0.0	0.0	0.0
62.5	EVO1-0hr-62.5mg/L	96.1	79.4	64.9	82.5
62.5	EVO1-96hr-62.5mg/L	74.7	59.0	4.9	55.7
125	EVO1_0hr-125mg/L	153.1	130.4	109.8	134.5
125	EVO1_96hr-125mg/L	144.2	118.4	13.5	110.5
250	EVO1_0hr-250mg/L	296.0	248.2	203.1	256.5
250	EVO1_96hr-250mg/L	302.5	248.6	31.8	232.4
500	EVO1_0hr-500mg/L	569.8	483.0	377.3	494.4
500	EVO1_96hr-500mg/L	539.7	493.9	55.7	440.9
1000	EVO1_0hr-1000mg/L	1099.1	961.0	754.9	973.4
1000	EVO1_96hr-1000mg/L	808.0	981.2	100.0	789.6
0	EVO2-0hr-0mg/mL	0.0	0.0	0.0	0.0
0	EVO2-48hr-0mg/mL	0.0	0.0	0.0	0.0
1.25	EVO2-0hr-1.25mg/L	3.3	1.1	0.0	1.4
1.25	EVO2-48hr-1.25mg/L	3.0	1.2	0.0	1.4
2.5	EVO2-0hr-2.5mg/L	4.6	2.4	1.1	2.9
2.5	EVO2-48hr-2.5mg/L	4.8	2.4	0.5	2.9
5	EVO2-0hr-5mg/L	7.6	4.7	3.4	5.4
5	EVO2-48hr-5mg/L	7.1	4.7	2.0	5.0
10	EVO2-0hr-10mg/L	12.9	9.7	7.6	10.4

10	EVO2-48hr-10mg/L	12.5	9.6	1.8	9.4
20	EVO2-0hr-20mg/L	25.4	19.4	17.2	21.0
20	EVO2-48hr-20mg/L	25.5	20.1	4.6	19.5
	EV0-4 -0hr -B1	0.0	0.0	0.0	0.0
	EV0-4- 0hr -PC1	0.0	0.0	0.0	0.0
	EV0-4-0hr-TC1	3.5	0.2	0.5	1.3
	EV0-4- 0hr -TS1	7.2	0.0	0.0	2.0

 

It was determined that the limit of detection (LOD) of benzylated polyamine peaks were 1.1 µg/mL for Peak 1 (see std 2, and 0.55 µg/mL for peaks 2 and 3 (see std 1), while the limit of quantifications (LOQ) were 5 µg/mL for peak 1 (see std 3) and 1.1 µg/mL for peak 2 and 3 (see std 2).

Validity criteria fulfilled:

yes

Conclusions:

Effect of benzylated polyamine on the growth rate of Freshwater Alga was examined using OECD 201: 2006 test method. Determined IC10 endpoint was 0.4457 mg/L with 0.3445 and 0.6129 mg/L 95% confidence limits. The IC50 endpoint was determined to be 2.7662 mg/L with 2.5607 and 2.9628 mg/L 95% confidence limits.

Executive summary:

Effect of benzylated polyamine on the growth rate of  Freshwater Alga was examined using OECD 201: 2006 test method. Determined IC10 endpoint was 0.4457 mg/L with 0.3445 and 0.6129 mg/L 95% confidence limits. The IC50 endpoint was determined to be 2.7662 mg/L with 2.5607 and 2.9628 mg/L 95% confidence limits.

Description of key information

Effect of benzylated polyamine on the growth rate of  Freshwater Alga was examined using OECD 201: 2006 test method. Determined IC10 endpoint was 0.4457 mg/L with 0.3445 and 0.6129 mg/L 95% confidence limits. The IC50 endpoint was determined to be 2.7662 mg/L with 2.5607 and 2.9628 mg/L 95% confidence limits.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.766 mg/L

EC10 or NOEC for freshwater algae:

0.446 mg/L