Sam ple Processing M ethods
Sam ple Processing M ethods
m in im um n um ber of in vertebrates for iden tification . L evel 1 A ssessm en ts
T h e m ain objective is to group th e in vertebrates by L evel 1 assessm en ts follow a sim plified sam ple
order an d determ in e th e n um ber of sen sitive or processin g procedure com pared to L evel 2 or
toleran t taxa presen t (see th e A nalysis & E valuation
Stream Macroinvertebrate Protocol
12 - 6
Water Quality Monitoring Guidebook
Section below for a discussion of “ sen sitive” an d “ toleran t” taxa). A s a result, L evel 1 studies h elp
• L abeling tape and alcohol-resistant marking
pens
volun teers recogn ize th e im portan ce of th e in vertebrate com m un ity as in dicators of a stream ’ s
• Forceps
con dition s an d provide a gen eral in dication of disturban ce.
• Squirt B ottle & plastic spoon • T ally counter (optional)
K ey E lem ents
1. R em ove all invertebrates from sam ples
Procedure
collected w ithin the sam e habitat at the sam e • T o sort the sample, place the composited reach.
sam ple into the m esh bottom ed sorting tray.
2. Sort specim ens into individual containers (ice
D E Q uses the equipm ent described by C aton cube trays are often used) by order: M ayflies,
Stoneflies, C addisflies, etc. • Place the mesh bottomed tray into the plastic
3. V isually estim ate the num ber of different types outer tray and add approxim ately 3 cm of w ater of taxa w ithin each order. For exam ple, how
to facilitate the even distribution of debris. In m any different looking m ayflies are there?
the field, place the tray on a level surface or tripod platform .
4. R ecord the num ber of different taxa w ithin each order and count how m any are present.
E venly distribute the material in the tray and lift the m esh bottom tray out of the w ater.
B ased on the num bers recorded, a general w ater quality rating can be calculated as described in the
• T he sorting tray is divided into thirty separate
A n a ly sis a n d E va lu a tio n Section.
6 X 6 cm squares. U se the random num ber table to select a m inim um of four of these squares. U se the 6 X 6 square sorting device
L evel 2 and 3 A ssessm ents (included in subsam pling tray kit recom m ended T he goal of the sam ple processing procedures for
by C aton) to isolate the four square and rem ove L evel 2 and 3 studies is to create an unbiased,
the selected m aterial.
random representative subsam ple of m acroinvertebrates from the com posited stream
D istribute the contents of the four squares into bottom sam ple of debris.
a separate w hite plastic tray w ith a sm all T he size of the subsam ple is a m inim um of 300
quantity of clean w ater. A ll the individuals. T he sam e size subsam ple should be
m acroinvertebrates are rem oved w ith forceps used for all sites for effective com parisons.
and placed in a labeled vial of alcohol. A n inside paper and pencil label is recom m ended
as w ell as an exterior label. • Subsampling tray (see C aton, 1991) and
E quipm ent
A minimum of 300 specimens and four squares associated sorting equipm ent
are sorted. If necessary, an additional one or m ore squares m ust be sorted to attain the
• T ripod w ith sorting tray platform for field 300 organism m inim um sam ple size. A ll
sorting (optional) organism s are com pletely rem oved from all • R andom number table, or other random number
sub-sam pled squares to avoid biasing the generator
m acroinvertebrate sam ple tow ard the larger, m ore visible species. U se a tally counter for
D enatured ethanol best results. K eep track of the num ber of • squares subsam pled in order to estim ate the V ials, approximately 20 mls.
original m acroinvertebrate density in the approach (costs are typically $50 to $75 per stream .
sam ple). Four im pairm ent categories m ay be • discerned at this level: non- im paired, slightly-
T he C aton sorting tray has thirty squares, each im p a ired , m oderately- im paired, and six centim eters square. W hen four D -fram e se v e re ly -im p a ired . T able 12-3 show s the kick sam ples are com posited, each square recom m ended level of taxonom y for each order. represents approxim ately sixty square
L evel 2 and 3 Identification M ethods Identifying Invertebrates
centim eters of stream bottom .
E quipm ent
M ethod overview
D issecting microscope (10X -60X zoom) T hree different levels of “ taxonom ic identification”
• L ight source
can be used after specim ens are sorted: order,
fa m ily or genus/species level. T he level of
• Forceps
taxonom ic identification is im portant in determ ining the cost and expertise needed for the analysis, as
• Petri dish
w ell as the resolution and sensitivity of the data to • detect environm ental im pacts. M acroinvertebrate taxonomic keys. See references for recom m ended keys (keys in bold Level 1 assessm ents do not identify organism s
type are the m ost im portant) beyond the order level (E phem eropter, Plecoptera,
D iptera, etc.). W ithin each order organism s are
D ata recording form
sim ply lum ped into sim ilar looking groups. T his
Procedure
approach is useful for dem onstrating the variety of organism s living in a stream reach, but has lim ited
• If the sample w as not sorted in the field, then value in assessing differences betw een sites. In
lab sort according to the procedure described in general a rough approxim ation of the invertebrate
the Sam ple Processing M ethods section com m unity can be determ ined and sam ple sites
(above).
categorized as having either an adequate or lim ited • Identify the macroinvertebrates to the invertebrate com m unity. Further sam pling and
taxonom ic level desired. T able 12-3 lists the m ore detailed analysis should be perform ed using
level of taxonom ic identification for different L evel 2 or L evel 3 assessm ent m ethods if concerns
m acroinvertebrate groups recom m ended for about a stream ’s condition exist.
L evel 3 assessm ents.
Level 2 assessm ents rely on fam ily level identification for assessing the invertebrate
• Identification to genus/species should be com m unity. Fam ily level identification is faster
perform ed by experienced entom ologists using current taxonom ic keys (see Taxonom ic
and requires less expertise than genus/species level, but is less sensitive. T hree levels of
R eferen c e s ) under the supervision of a senior aquatic entom ologist. Fam ily level
biological conditions m ay be determ ined from identification is possible by less experienced
fa m ily level identification: non-im paired, staff, but sufficient taxonom ic training is still m o d era tely im p a ired , and severely im paired.
critical.
L evel 3 assessm ents rely on genus/species identification for m ost orders. T his is the m ost
• T he number of each taxon is noted on a tally sheet along w ith other site identifier
effective level for evaluating stream conditions and inform ation (see D ata R ecording Form s). evaluating differences betw een sites. It also
requires the m ost tim e and expertise. B ecause of • Q uality control procedures described in the Q uality the identification skills required, contracting
A ssu ra n ce section (see below and C hapter 4 D ata specim en identification to a qualified taxonom ist
Q u a lity ) should be com pleted to evaluate the for L evel 3 assessm ents is often the m ost effective
quality of the sam ple identification.
• data are outlined in the A nalysis and E valuation T he biom etrics and biological condition
section.
assessm ents used to analyze the m acroinvertebrate
T able 12-3. L evel of m acroinvertebrate identification for L evel III analysis.
L evel of Identification
T axon
G enus S pecies A m phipoda
O rder
F am ily
S ub-fam ily
X X (scuds) A rachnida
(spider and w ater m ites) C oleoptera (m ost beetles)
E lm idae X X (riffle beetles) D iptera (m ost true flies)
X C hironom idae
(m idges) E phem eroptera
X X (m ayflies) G astropoda
X (snails) H em iptera
S om e
X (true bugs e.g. w ater boatm en) L epidoptera
X (butterflies & m oths) M egaloptera
X (hellgarm m ites & alder flies) O donata
X (dragonflies & dam selflies) O ligochaeta
S om e
(w orm s) O stracoda
(seed shrim p) P elecypoda
(clam s) P lecoptera
X X (stoneflies) T richoptera
S om e
X (caddis flies) T urbellaria
(flatw orm s) H irudinea
(leeches)
• M ayflies (E phemeroptera) O verview
A nalysis & E valuation
• Stoneflies (Plecoptera)
D ata analysis and evaluation of stream conditions are often based on assessing the characteristics of
C addisflies (T richoptera) the m acroinvertebrate com m unity. T his is often
accom plished through the use of “ m etrics.” M etrics T o lera n t organism s are those that tolerate high are m easures of com m unity characteristics based on
levels of disturbance and pollution, and rem ain single or m ultiple taxa. T he m etrics used in this
present after other groups have disappeared. T his m anual have been selected because they are know n
includes the follow ing orders: to change as a result of anthropogenic (hum an
A quatic w orms (O ligocheata) caused) disturbance. E xam ples include total taxa
richness, m ayfly richness, % dom inant taxa, etc.
• L eeches (H irudinea)
E ach m etric is scored (usually 1, 3, or 5) based on
B lackflies (D iptera)
scoring criteria. A ll the individual m etric scores are then sum m ed together for an overall “ B iotic
• M idges (D iptera)
Index” score for the site. T he final biotic index
• Snails (G astropoda)
falls w ithin a know n range indicating different levels of im pairm ent.
Since L evel 1 assessm ents are prim arily an educational level, different levels of stream
C riteria for the individual m etric scores and the im pairm ent cannot be calculated. T he generalized im pairm ent categories for the biotic index scores
data only provide enough inform ation to determ ine are based on data collected from reference sites in
w hether the m acrovinvertebrate com m unity appears regions sim ilar to the study sites being evaluated.
to be adequate or lim ited. Sites w here each of the T he m etric values presented here are based on
three sensitive orders (m ayflies, stoneflies, and reference site data collected by the D epartm ent of
caddisflies) are present and tolerant organism s such
E nvironm ent Q uality (D E Q ) in the O regon C oast as w orm s, leeches and blackflies m ake up less than R ange. T hese criteria w ill w ork for assessing riffle
50% of the total organism s counted from the sam ples from other O regon coastal stream s, but
sam ple are considered adequate. If any one of the should not be used to assess other habitat types or
three sensitive orders are absent and/or tolerant stream s from other areas of the state. T he m entors
organism s equal m ore than 50% of the total in the listed at the beginning of this section should be
sam ple, the site has a lim ited invertebrate contacted for assessing stream s outside the coast
com m unity. L evel 2 or 3 assessm ents are then range for the m ost appropriate m etric criteria.
necessary to evaluate the sites further. L evel 1 A ssessm ents
T o develop a general evaluation of a site w ith L evel
L evel 2 A ssessm ents
1 data the invertebrates are first separated by order, L evel 2 site assessm ents are based on fam ily level then the num ber of different “ looking” organism s in
identifications. T he num ber of organism s in each each order are recorded and counted. T he different
fa m ily are counted and recorded. T hese values are orders of invertebrates can be generally classed as
then used to determ ine m etric values or scores. “ sensitive,” or “ tolerant.”
M etric scores are sum m ed to determ ine the overall rating for the site. T he follow ing table outlines the
S en sitive organism s are those m ost sensitive to fam ily level m etrics and scoring criteria. pollution and are first to disappear from the
T axa R ichness
invertebrate com m unity as a result of disturbance T his is the total num ber of invertebrate fam ilies or pollution. T hose considered sensitive include the
identified from the sam ple.
follow ing:
Stream Macroinvertebrate Protocol
12 - 10
Water Quality Monitoring Guidebook Water Quality Monitoring Guidebook
100.
T his is the total num ber of m ayfly fam ilies identified from the sam ple.
% D om inance (top 3 taxa) T his is the total num ber of the three m ost abundant
Stonefly R ichness organism s divided by the total num ber sorted from
T his is the total num ber of stonefly fam ilies the sam ple, m ultiplied by 100. identified from the sam ple.
A dd up the scores for each m etric to determ ine the T his is the total num ber of caddisfly fam ilies
C addisfly R ichness
total site score or biotic index. T he total scores are identified from the sam ple.
used to determ ine three levels of im pairm ent as % C hironom idae
indicated below .
T his is the total num ber of chironom ids (m idges) in the sam ple divided by the total num ber of
T ab le 12-4. F amily level metrics an d scorin g criteria.
Scorin g C riteria
Score M etric
R aw
5 3 1 (C ircle) T axa R ich n ess
V alu e
5 3 1 M ayfly R ich n ess
5 3 1 Ston efly R ich n ess
0 5 3 1 C addisfly R ich n ess
>3
1 -3
5 3 1 % C h iron om idae
5 3 1 (T op 3 T axa)
Score R an ge
Stream C on dition
>23 N o im p airm en t: p asses L evel 2 assessm en t. In dicates good diversity of in verteb rates an d stream con dition s w ith little distu rb an ce. Fu rth er sam p lin g w ill h elp con firm th e site’ s con dition as u n im p aired.
1 7 -2 3 M oderate Im p airm en t: eviden ce of som e im p airm en t exists. R eq u ires fu rth er stu dy an d m ore detailed an alysis.
<17 Severe Im p airm en t: fails L evel 2 assessm en t. E viden ce of stream distu rb an ce exists. Fu rth er stu dy m ay b e w arran ted to con firm level of im p airm en t an d p oten tial cau ses.
Stream Macroinvertebrate Protocol
12 - 11
Water Quality Monitoring Guidebook
Stream Macroinvertebrate Protocol
12 - 12
Water Quality Monitoring Guidebook
L evel 3 A ssessm ents L evel 3 assessm ents are based on genus/species
level identifications, w hich provides a m ore sensitive m easure of the invertebrate com m unity’s condition. T w o analytical approaches can be used for L evel 3 assessm ents: m ultim etric analysis, or m u ltiva ria te analysis. T o m ake accurate assessm ents betw een sites, using either m ultim etric or m ultivariate analysis techniques, the sam e level o f id en tifica tio n m u st b e u sed fo r ea ch ta x o n o m ic
g ro u p fo r a ll sites b e in g c o m p a re d .
B ecause levels
of identification can vary betw een taxonom ists or betw een sites due to m aturity of specim ens or preservation quality, each data set should be checked by a taxonom ist for identification consistency.