Appendix Part 2 Aquatic toxicity tests, Danish Environmental Protection Agency

Environmental and Health Assessment of Substances in Household Detergents and Cosmetic Detergent Products

Appendix Part 2 Aquatic toxicity tests

Algae growth inhibition test.

The toxicity of the test substances to the growth of the micro algae (Pseudokirchneriella subcapitata formerly Selenastrum capricornutum) was determined in a 72 hour test according to ISO 8692 guideline (ISO 1989). Exponentially growing batch cultures of algae were exposed to a serial dilution of the test substance. The test was performed with 3 replicate batches at each concentration and 6 control batches. The test was incubated on a shaking table under constant temperature and light. The cell densities were determined by fluoremetric measurement after 24, 48, and 72 hours of exposure. The inhibition of growth at each test concentration was determined as the average growth rate in per cent of the control growth rate. The concentrations, which inhibited the growth rate 10 and 50%, were calculated by use of a computer program ‘Toxedo’ (VKI 1992). The highest concentration at which no significant effect was observed (No Observed Effect Concentration, NOEC), was determined by Dunnett’s procedure (US-EPA 1989).

Daphnia, acute immobilization test.

The toxicity of the test substances was tested in a 48-hour immobilization test with Daphnia magna according to ISO 6341 guideline (ISO 1996). Groups of daphnids (4 replicates of 5 animals) were exposed to a serial dilution of the substance. The number of immobile animals was registered after 24 and 48 hours. Oxygen level, pH and temperature were determined at test start and termination of the test. On the basis of the test results, the concentrations, at which 10, 50 and 90% of the animals were immobilized (EC10, EC50 and EC90) were calculated by Probit analysis (Statens Naturvårdsverk 1992).

The results of the tests are summarized below:

Table A20
Effects of parabens to Pseudokirchneriella subcapitata and Daphnia magna. Detailed data are given in the following sections.

Test substance/species	EC50 (mg/l)	NOEC (mg/l)	Test duration
Methylparaben
Pseudokirchneriella subcapitata	91 (90-93)^A	20	72 h
Daphnia magna	11.2(5.7-22.0)	2.0	48 h
Ethylparaben
Pseudokirchneriella subcapitata	18 (17-19)	5.0	72 h
Daphnia magna	20-50	10	48 h
Propylparaben
Pseudokirchneriella subcapitata	15 (15-16)	5.0	72 h
Daphnia magna	15.4(8.0-32.3)	1.0	48 h

^{A Parentheses indicate 95% confidence intervals.
Inhibition of the growth of Pseudokirchneriella subcapitata with "
Ethylparaben".
Statistical parameters calculated from continuous responses based on continuous mean.

Testtype : Growth Inhibition Test.

6 doses and 16 responses have been used during calculations.
Control values.

Concentration

in mg/l
Growth
Inhibition

in per cent

Control 1
1.956
-

Control 2
1.998
-

Control 3
1.970
-

Control 4
2.017
-

Control 5
2.054
-

Control 6
2.004
-

Control mean
2.000
0

Experimental Data.

Concentration

in mg/l
Growth
Inhibition

in per cent

1
2.009
0.0

1
1.991
0.4

1
2.018
0.0

2
2.016
0.0

2
2.027
0.0

2
2.018
0.0

5
1.985
0.7

5
1.982
0.9

5
1.973
1.3

10
1.698
15.1

10
1.677
16.1

10
1.719
14.0

20
0.667
66.6

20
0.776
61.2

20
0.661
66.9

50
0.330
83.5

50
0.202
89.9

50
0.336
83.2

100
0.072
96.4

100
0.048
97.6

100
0.029
98.5

Dunnett’s procedure:
NOEC: 5 mg/l

LOEC: 10 mg/l
EC-values and limits of the 95% confidence interval.

y(EC)
LCL
EC(yo)
UCL

10
6
6
7

50
17
18
19

90
45
49
53

Inhibition of the growth of Pseudikirchneriella subcapitata with
"Methylparaben".
Statistical parameters calculated from continuous responses based on continuous mean.

Testtype : Growth Inhibition Test.

6 doses and 13 responses have been used during calculations.
Control values.

Concentration

in mg/l
Growth
Inhibition

in per cent

Control 1
2.102
-

Control 2
2.094
-

Control 3
2.101
-

Control 4
2.123
-

Control 5
2.118
-

Control 6
2.102
-

Control mean
2.107
0

Experimental Data.

Concentration

in mg/l
Growth
Inhibition

in per cent

2
2.046
2.9

2
2.129
0.0

2
2.086
1.0

5
2.129
0.0

5
2.109
0.0

5
2.093
0.6

10
2.151
0.0

10
2.132
0.0

10
2.029
3.7

20
2.108
0.0

20
2.114
0.0

20
2.137
0.0

50
1.836
12.8

50
1.820
13.6

50
1.779
15.6

100
0.917
56.5

100
0.908
56.9

100
0.908
56.9

200
0.337
84.0

200
0.337
84.0

200
0.341
83.8

Dunnett’s procedure:
NOEC: 20 mg/l

LOEC: 50 mg/l
EC-values and limits of the 95% confidence interval.

y(EC)
LCL
EC(yo)
UCL

10
29
31
32

50
90
91
93

90
> 200

Inhibition of the growth of Pseudokirchneriella subcapitata with "
Propylparaben ".
Statistical parameters calculated from continuous responses based on continuous mean.

Testtype : Growth Inhibition Test.

6 doses and 14 responses have been used during calculations.
Control values.

Concentration

in mg/l
Growth
Inhibition

in per cent

Control 1
2.070
-

Control 2
2.066
-

Control 3
1.972
-

Control 4
2.043
-

Control 5
2.046
-

Control 6
2.060
-

Control mean
2.043
0

Experimental Data.

Concentration

in mg/l
Growth
Inhibition

in per cent

1
2.062
0.0

1
2.039
0.2

1
2.056
0.0

1
2.090
0.0

1
2.072
0.0

1
2.058
0.0

2
2.033
0.5

2
2.063
0.0

2
2.055
0.0

5
1.992
2.5

5
1.976
3.3

5
1.966
3.8

10
1.675
18.0

10
1.625
20.5

10
1.661
18.7

20
0.529
74.1

20
0.596
70.8

20
0.522
74.4

50
0.070
96.6

50
0.085
95.8

50
0.283
86.1

Dunnett’s procedure:
NOEC: 5 mg/l

LOEC: 10 mg/l
EC-values and limits of the 95% confidence interval.

y(EC)
LCL
EC(yo)
UCL

10
7
7
8

50
15
15
16

90
30
32
35

Primary data for acute
test

TEST ORGANISM:
Daphnia Magna
LAB. NO.:
8119/365

TEST COMPOUND:
Ethylparaben
FILE NO.:
11166

START DATE:
1999.09.07
OBSERVERS:
HeE

pH
O₂-content (%
saturation)

START
END
START
END

Control
7.8
7.8
99
97

Highest concentration
7.9
7.9
> 100
95

Concentration
Total
No. of anim.
NUMBER OF
IMMOBILE

mg/l
number of
per vessel
24 HOURS
48 HOURS

animals per
A
C
E
A
C
E
Total
A
C
E
Total

conc.
B
D
F
B
D
F

B
D
F

Control
30

5
5
5
0
0
0
0

0
0
0
0

5
5
5
0
0
0
0
0
0

1
20

5
5

0
0

0

0
0

0

5
5

0
0

0
0

2
21

5
5

0
0

0

0
0

0

5
6

0
0

0
0

5
20

5
5

0
0

0

0
0

0

5
5

0
0

0
0

10
20

5
5

0
0

0

0
0

0

5
5

0
0

0
0

20
20

5
5

0
0

0

2
4

14

5
5

0
0

4
4

50
20

5
5

5
5

20

5
5

20

5
5

5
5

5
5

100
20

5
5

5
5

20

5
5

20

5
5

5
5

5
5

Ethylparaben

mg/l
0 HOURS
24 HOURS
48 HOURS

Conc.
O₂
pH
O₂
pH
O₂
pH

Control
99
7.8
-
-
97
7,8

20
> 100
7.9
-
-
95
7,9

50
> 100
7.9
98
7,8
-
-

100
> 100
7.9
97
7,8

O₂-contents are given in % of saturation

24 hours
48 hours

E10
between
20 - 50
between
10-20

E50
20 - 50
20-50

E90
20 - 50
20-50

Unit: mg/l

Primary data
for acute test

TEST ORGANISM:
Daphnia magna
LAB. NO.:
81119/366

TEST COMPOUND:
Methylparaben
FILE NO.:
11166

START DATE:
1999.09.14
OBSERVERS:
CS

pH
O₂-content
(% saturation)

START
END
START
END

Control
7.7
7.9
100
100

Highest concentration
7.6
7.7
100
100

Concentration
Total
No. of anim.
NUMBER OF
IMMOBILE

mg/l
number of
per vessel
24 HOURS
48 HOURS

animals per
A
C
E
A
C
E
Total
A
C
E
Total

conc.
B
D
F
B
D
F

B
D
F

Control
30
5
5
5
0
0
0
0
0
0
0
0

5
5
5
0
0
0

0
0
0

2.0
20
5
5

0
0

0
0
0

0

5
5

0
0

0
0

5.0
20
5
5

0
0

0
0
0

1

5
5

0
0

0
1

10
20
5
5

2
2

7
3
2

10

5
5

1
2

2
3

20
20
5
5

3
1

11
5
4

19

5
5

5
2

5
5

50
20
5
5

3
3

11
5
5

18

5
5

3
2

5
3

100
20
5
5

5
5

20
5
5

20

5
5

5
5

5
5

200
20
5
5
*
5
5

20
5
5

20

5
5

5
5

5
5

* Dead after 10 minutes

Methylparaben

mg/l
0 HOURS
24 HOURS
48 HOURS

Conc.
O₂
pH
O₂
pH
O₂
pH

Control
100
7.7
-
-
100
7.9

50
100
7.6
-
-
100
8.0

100
100
7.6
100
7.6
-
-

200
100
7.6
100
7.7
-
-

O₂-contents are given in % of saturation

LOG
24 hours
48 hours

EC10
6.24

(0.88 - 12.5)
4.50

(0.56 – 7.80)

EC50
22.4

(10.6 – 47.6)
11.2

(5.73 – 22.0)

EC90
80.7

(40.1 - > 200 )
27.8

(16.0 - > 200)

Unit: mg/l

Primary data
for acute test

TEST ORGANISM:
Daphnia Magna
LAB. NO.:
8119/367

TEST COMPOUND:
Propylparaben
FILE NO.:
11166

START DATE:
1999.09.15
OBSERVERS:
CS

pH
O₂-content
(% saturation)

START
END
START
END

Control
7.7
7.6
100
100

Highest concentration
7.7
7.6
100
100

Concentration
Total
No. of anim.
NUMBER OF
IMMOBILE

mg/l
number of
per vessel
24 HOURS
48 HOURS

animals per
A
C
E
A
C
E
Total
A
C
E
Total

conc.
B
D
F
B
D
F

B
D
F

Control
30

5
5
5
0
0
0
0

0
0
0
0

5
5
5
0
0
0
0
0
0

0.5
20

5
5

0
0

0

0
0

0

5
5

0
0

0
0

1
20

5
5

0
0

0

0
0

0

5
5

0
0

0
0

2
20

5
5

0
1

1

0
1

1

5
5

0
0

0
0

5
20

5
5

0
0

0

0
0

0

5
5

0
0

0
0

10
20

5
5

0
0

0

0
0

0

5
5

0
0

0
0

20
20

5
5

1
1

4

4
4

18

5
5

0
2

5
5

50
20

5
5

5
5

20

5
5

20

5
5

5
5

5
5

100
20

5
5

5
5

20

5
5

20

5
5

5
5

5
5

Propylparaben

mg/l
0 HOURS
24 HOURS
48 HOURS

Conc.
O₂
pH
O₂
pH
O₂
pH

Control
100
7.7
-
-
100
7.6

2
100
7.7
-
-
100
7.6

50
100
7.7
100
7.6
-
-

100
100
7.7
100
7.6
-
-

O₂-contents are given in % of saturation

LIN
24 hours
48 hours

EC10
15.2

(5.70 - 22.9)
9.57

(• - 15.0)

EC50
27.5

(20.3 – 45.8)
15.4

(7.97 – 32.3)

EC90
39.8

(29.6 – 74.0)
21.3

(15.7 – 74.9)

Unit: mg/l

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