Registration Dossier

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009.08.13 to 2009.09.15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study Conducted According to GLP and OECD 210, Fish, Early-life Stage Toxicity Test Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material as cited in test report: SLI (76) Stripped
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Nominal Concentrations: 0.32, 0.56, 1.0, 1.8 mg/L
- Measured Total Concentrations: 0.11, 0.22, 0.36, 0.58 mg/L
- Measured Dissolved Concentrations: 0.045, 0.11, 0.20, 0.33 mg/L

- Sampling method (total concentration):
1. 10 ml of sample taken by syringe (Plastic 10ml Luer-Lok syringe)
2. 5.0 ml expressed to waste
3. Remaining 5.0 ml collected in a glass centrifuge tube
4. 5.0 ml of methanol added to the centrifuge tube using the same syringe
5. Sample thoroughly mixed and ultra-sonicated for 15 minutes
6. Sample centrifuged at 960 g for 30 minutes at 22 °C
7. Aliquot of the supernatant taken by for analysis

- Sampling method (dissolved concentrations)
1. 10 ml of sample taken by syringe (Plastic 10ml Luer-Lok syringe)
2. Luer lock syringe filter (Millex – HV, Durapore PVDF, 25mm, 0.45µm) attached to syringe
3. 5.0 ml filtered to waste
4. Remaining 5.0 ml filtered into a glass centrifuge tube
5. Filter discarded
6. 5.0 ml of methanol added to the centrifuge tube using the same syringe
7. Sample thoroughly mixed and ultra-sonicated for 15 minutes
8. Sample centrifuged at 960 g for 30 minutes at 22 °C
9. Aliquot of the supernatant taken by for analysis

- Sample storage conditions before analysis: No data

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow
- Source: embryos used in this study (batch 167-09) were obtained from brood stock held at Brixham Environmental Laboratory
- Source: adult fish used in this study (batch 207-08) were received, as embryos, at Brixham Laboratory from Aquatic Research Organisms, PO Box 1271, 1 Lafayette Road, Hampton, NH 03842, USA.

During the months prior to the start of the exposure they showed no evidence of disease and were held at a temperature of 25 ± 1 degrees C.

The broodstock used for the study were held in 45 l glass tanks under artificial lighting and were contained in water as described for the test dilution water, except that the water was only filtered to 10 µm before use. They were fed daily using a proprietary brand of high protein pelleted fish food, supplemented with frozen brine shrimp. No mortalities were recorded within seven days prior to the start of the study.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
The adult fish were separated into breeding groups with approximately 3 males and 6 females in each tank. The eggs from the spawnings in 6 brood tanks were collected and pooled in a dish filled with dilution water. Each batch of eggs was less than 24 hours old, and all batches were at either early-cleavage or morula stage of development. Sets of 5 eggs were impartially selected, microscopically examined for viability and placed into incubation cups. This process was repeated until each incubation cup contained a nominal 15 impartially selected eggs.

POST-HATCH FEEDING
- Start date: 2009-08-17
- Type/source of feed: rotifers for first 4 days followed by rotifers and artemia
- Amount given: 0-7 d: 4.5 mL rotifers per tank per feed; 5-26 d: 0.30 to 4.5 mL artemia (increasing amounts during study period)
- Frequency of feeding: twice per day

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Post exposure observation period:
No data

Test conditions

Hardness:
Measured once per week in all test concentrations and control vessels. Reported total hardness at start of study was 35.1 (SD = 1.1) mg/L CaCO3 and at the end of the study was 50.3 (SD = 1.1) mg/L CaCO3.
Test temperature:
25.0 to 25.7 degrees C
pH:
7.43 to 7.80
Dissolved oxygen:
7.8 to 9.0 mg/L
Salinity:
0.1
Nominal and measured concentrations:
- Nominal Concentrations: 0.32, 0.56, 1.0, 1.8 mg/L
- Measured Total Concentrations: 0.11, 0.22, 0.36, 0.58 mg/L
- Measured Dissolved Concentrations: 0.045, 0.11, 0.20, 0.33 mg/L
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume):
Egg incubation cups (2 per replicate tank) were made from 80 mm standard lengths of 50 mm diameter glass tubing with nylon mesh attached to the bottom of each cup using silicone sealant. The cups were suspended in the test vessels and oscillated vertically over a distance of approximately 20 - 50 mm at a rate of 2 oscillations min-1.
- Test vessel:
The test vessels were of all glass construction, rectangular in shape with external dimensions of 305 mm x 205 mm x 210 mm (standard length x width x depth) and a maximum capacity of approximately 12 L. The test solution volume used was nominally 9.5 L. Two replicate test vessels (termed A and B) were employed for each treatment.

- Type of flow-through (e.g. peristaltic or proportional diluter):
A dynamic (flow through) test system was used for the study. The test apparatus was constructed of glass with a minimum of other materials (silicone tubing and sealant) in contact with the test solutions. Settling vessels, 2 L glass beakers with a nominal working volume of 1000 ml, were introduced to remove as much particulate matter from the system prior to the test solution reaching the test vessels.
- Renewal rate of test solution (frequency/flow rate): 35 tank volumes per day
- No. of fertilized eggs/embryos per vessel: 2x15 per vessel
- No. of vessels per concentration (replicates): 2 tanks
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The dilution water was dechlorinated tap water, which had been passed through activated carbon, coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. Salts were added, as required, to maintain minimum hardness levels, and the treated water was then passed through an ultraviolet steriliser to a set of 20 and 10 µm filters. The supply was then delivered to a temperature controlled header tank in the test laboratory set to the nominal test temperature of 25 ± 1 degrees C and finally re-filtered at 5 µm before use.
- Dissolved organic carbon: 0.440 mg/L
- Suspended solids: <3.0 mg/L
- Metals:

Metals reported as µg/L (sampled on 3 June 2009)

Cd Hg Ag Al As Cr Co
<0.100 <2.000 <1.00 <10.0 <1.00 <0.500 <1.00

Cu Fe Pb Mn Ni Zn B
<1.000 <30.0 <2.000 <10.0 <1.00 <5.00 <100.

- Pesticides:

Pesticides, highest values reported as µg/L (sampled on 3 June and 16 September# 2009)

Organochlorine pesticides Organophosphorous pesticides PCBs
<0.0500 <0.0100 <0.00500

- Chlorine: <2 µg/L
- Alkalinity: 22.9 mg/L
- Ca/Mg ratio: 11.2:3.0
- Conductivity:208 µS/cm
- Salinity: <0.1
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH and conductivity were measured daily. Alkalinity, hardness and free and residual chlorine measured once per week. All other water quality parameters were measured quarterly.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hrs light, 8 hrs dark (with a 20 minute dawn and dusk transition)
- Light intensity: 680, 740 and 540 lux taken from centre and either end of test rig.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Hatch, Survival, length and weight

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
A range finder study was conducted but not according to GLP and not included in this report.

POST-HATCH DETAILS
- Begin of post-hatch period: 2009-08-17
- No. of hatched eggs (alevins)/treatment released to the test chamber:
For each concentration and control there were 4 replicates of 15 eggs (a total of 60 eggs)
Control: 60 hatched
Dissolved measured
0.045 mg/L: 57 hatched
0.11 mg/L: 60 hatched
0.20 mg/L: 58 hatched
0.33 mg/L: 3 hatched
- Release of alevins from incubation cups to test chamber on day no.: 24 h after hatch day

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 60 x 5 (300)
- Removal of eggs to check the embryonic development on day no.: no data
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
number hatched
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.045 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
length
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.045 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.33 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
number hatched
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.58 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 0.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
length
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.11 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
>= 0.22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
- Mortality/survival at embryoand larval stages (see table 1 below)
- Days to hatch or time to release of young: 4 days
- Numbers hatched (see table 1 below)
- Observations on body length and weight of young and/or exposed parents at one or more time periods (see table 2 and 3)
- Number of healthy fish at end of test - see table 1
- Type of and number with morphological and behaviour abnormalities:
Observations of the behaviour and any symptoms of abnormality were made throughout the test and the following were described:
Four of the newly hatched fry appeared to be paler and weaker than the rest (nominal 0.32, 0.56 & 1.0 mg/L). On Day 7 post hatch two smaller, weaker fry were noted as well as 2 deformed fry (nominal 0.56 mg/L). By Day 14 post hatch six small fry were observed within the study (nominal 0.32, 0.56 & 1.0 mg/L) and on Day 21 post hatch five small fry were observed (DWC and nominal 0.32, 0.56 & 1.0 mg/L). It was also noted that a number of the fry appeared to be paler in appearance to the rest (not concentration related). At the end of the study, Day 28 post hatch, 4 of the small fry remained (DWC and nominal 0.32, 0.56 & 1.0 mg/L) but there were no longer any pale or deformed fry present and the remainder of the fry appeared to be healthy.
- Effect concentrations exceeding solubility of substance in test medium: no data
- Incidents in the course of the test which might have influenced the results: none
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The acceptability of the test was considered from the Relative Standard Deviation (RSD), of the dry weights of the fish, which were alive at the end of the test in the controls. The values obtained were 34 and 25 % for A and B replicates, respectively. The RSD is defined as the standard deviation expressed as a percentage of the mean (this is equivalent to the coefficient of variation). The RSD for weight data were all less than 40% and are, therefore, considered acceptable according to the EPA Standard Evaluation Procedure.

Any other information on results incl. tables

Table 1: HATCH AND SURVIVAL DATA

 

Nominal conc of

SLI (76) STRIPPED

Mean measured conc of

SLI (76) STRIPPED

Dissolved

(Total)

 

(mg l-1)

Replicate

Number of eggs at start

Number of larvaea

Percentage hatch

Number of larvae surviving at

Percentage survival of larvae from release to 28 days post hatch

 

 

 

Hatcheda

Releasedb

Individual

Pooled

28 days

Individual

Pooled

 

 

 

(mg l-1)

 

 

 

 

replicates

replicates

post hatch

replicates

replicates

Dilution

water

control

Dilution

water

control

A1

15

15

15

100

100

28

 

93

 

 

90

 

 

 

A2

15

15

15

100

B1

15

15

15

100

26

 

87

 

B2

15

15

15

100

0.32

0.045

(0.11)

A1

15

14

14

93

95

24

 

83

 

 

81

 

 

 

A2

15

15

15

100

B1

15

13

13

87

22

 

79

 

B2

15

15

15

100

0.56

0.11

(0.22)

A1

15

15

15

100

100

21

 

70

 

 

73*

 

 

 

A2

15

15

15

100

B1

15

15

15

100

23

 

77

 

B2

15

15

15

100

1.0

0.20

(0.36)

A1

15

15

15

100

97

17

 

59

 

 

67*

 

 

 

A2

15

14

14

93

B1

15

14

14

93

22

 

76

 

B2

15

15

15

100

1.8

0.33

(0.58)

A1

15

1

0

7

5*

0

 

-

 

50*

A2

15

0

0

0

B1

15

0

0

0

1

50

B2

15

2

2

13

a: Live fry hatched

b: Surviving fry released into tank;boldfigures indicate an incubation cup in which there were fry which were dead when hatched (numbers of deaths are by subtraction from the number hatched)

*: Statistically significant difference between treatment and DWC (p <0.05)

Table 2 LARVAL STANDARD LENGTHS (mm) AT 28 DAYS POST-HATCH

Pooled replicates

 

Nominal conc of

SLI (76) STRIPPED

 

 

 

 

(mg l-1)

Mean measured conc of

SLI (76) STRIPPED

Dissolved

(Total)

 

(mg l-1)

Number of fish

Mean

 

 

 

Minimum

 

 

 

Maximum

 

 

 

Standard

deviation

Relative standard deviation

(%)

Dilution water control

Dilution water control

54

20.18

10.34

23.94

2.32

11

0.32

0.045

(0.11)

46

21.76

13.01

26.81

2.33

11

0.56

0.11

(0.22)

44

21.34

13.00

24.62

2.08

10

1.0

0.20

(0.36)

39

21.54

10.27

25.17

2.99

14

 The nominal 1.8 mg l-1concentration was not included in the calculations as only 1 larvae survived.

No significant differences were found.

Table 3: LARVAL DRY WEIGHTS (mg) AT 28 DAYS POST-HATCH

 

Nominal conc of

SLI (76) STRIPPED

 

(mg l-1)

Nominal conc of

SLI (76) STRIPPED

 

(mg l-1)

Number of fish

Mean

 

 

 

Minimum

 

 

 

Maximum

 

 

 

Standard

deviation

Relative standard deviation

(%)

Dilution water control

Dilution water control

54

31.81

1.90

51.00

9.54

30

0.32

0.045

(0.11)

54

31.66

5.10

65.70

16.75

30

0.56*

0.11*

(0.22)

44

37.44

6.80

58.50

10.84

29

1.0*

0.20*

(0.36)

39

39.14

2.50

62.10

14.02

36

 

* Statistically significant difference between treatment and DWC (p <0.05)

A statistically significant increase in dry weight was detected in the 0.56 and 1.0 mg l-1when compared with the dilution water control, however, this was not considered to be an adverse biological effect.

The nominal 1.8 mg l-1concentration was not included in the calculations as only 1 larvae survived.

Applicant's summary and conclusion

Conclusions:
For all the biological parameters included in the evaluation (hatch, survival, standard length, and dry weight) the overall results based on mean measured dissolved concentrations (numbers in brackets are mean measured total concentrations) are:
No Observed Effect Concentration (NOEC): 0.045 (0.11) mg/L
Lowest Observed Effect Concentration (LOEC): 0.11 (0.22) mg/L
Executive summary:

An OECD 210 Fish Early Life stage toxicity of SLI(76) stripped to fathead minnow was conducted.

For all the biological parameters included in the evaluation (hatch, survival, standard length, and dry weight) the overall results based on mean measured dissolved concentrations (numbers in brackets are mean measured total concentrations) are: No Observed Effect Concentration (NOEC): 0.045 (0.11) mg/L Lowest Observed Effect Concentration (LOEC): 0.11 (0.22) mg/L.